IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004 imex curation Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org 04-Aug-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) Regarding the origins of the Flag-Smad constructs; Supplementary Text states:- "Flag-Smad1, Flag-Smad2, Flag-Smad3, Flag-Smad4, and their deletion constructs have been described." Unfortunately it is not stated WHERE these constructs were described. It has been assumed these constructs are all of Human origin as the Addgene repository lists several Human Flag-Smad containing plasmids deposited by the last author - Joan Massague, but not specifically mentioning this publication. However, no other species is listed by Addgene for SMAD3 or 4 deposited by Joan Massague . As used in Supplementary Fig 2 the (murine) Flag-FoxG1 deletion constructs are referenced (Ref S2) to the Dou et al., 2000 publication. HA-FOXO3 (N terminal tag) (PMID:10102273 Sup Ref 1, Brunet et al., 1999) is Human in origin. HA-FOXO1 (N-terminal tag) (PMID:11696581 Sup Ref 10, Nakae et al., 2001) is Murine in origin. HA-FOXO4 (tag location unknown) (PMID:10217147 Sup Ref 6, Kops et al., 1999) is Human in origin. Myc-FOXG1 (tag location unknown) ( PMID:10938097 Sup Ref 2, Dou et al., 2000) is Murine in origin. Myc-Foxh1 (FAST 1/2) (N-terminal tag) (PMID:9858566) is Murine in origin. j-massague@ski.mskcc.org Accepted 2016-JUL-14 AT 17:06 BST AT 17:06 BST by PPORRAS Cell 2004 Only protein-protein interactions imex curation imex curation Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004 imex curation Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org 04-Aug-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro emsa supershift electrophoretic mobility supershift assay emsa supershift weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org Accepted 2016-JUL-14 AT 17:06 BST AT 17:06 BST by PPORRAS Cell 2004 Only protein-protein interactions imex curation imex curation Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004 imex curation Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org 04-Aug-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) It is stated that:- "The bacterial expression vectors pGEX4T1 encoding Smad domains fused to glutathione-S-transferase have been described (Seoane et al., 2001)." However this reference does not give the sequence of the SMAD constructs used in Supp.figure 2D -(MH1+ linker), so the approximate sequences have been taken from UniProt. j-massague@ski.mskcc.org Accepted 2016-JUL-14 AT 17:06 BST AT 17:06 BST by PPORRAS Cell 2004 Only protein-protein interactions imex curation imex curation Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004 imex curation Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org 04-Aug-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-hacat Homo sapiens immortalised keratinocytes hacat human immortalised keratinocytes anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) CoIP data using Smad2/3 was not recorded since the antibodies used were not specific enough to ascertain the protein identity. j-massague@ski.mskcc.org Accepted 2016-JUL-14 AT 17:06 BST AT 17:06 BST by PPORRAS Cell 2004 Only protein-protein interactions imex curation imex curation Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004 imex curation Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org 04-Aug-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) Regarding the origins of the Flag-Smad constructs; Supplementary Text states:- "Flag-Smad1, Flag-Smad2, Flag-Smad3, Flag-Smad4, and their deletion constructs have been described." Unfortunately it is not stated where these constructs were described. It has been assumed these constructs are all of Human origin as the Addgene repository lists several Human Flag-Smad containing plasmids deposited by the last author - Joan Massague, but not specifically mentioning this publication. However, no other species is listed by Addgene for SMAD3 or 4 deposited by Joan Massague . Previous work by the first author (Seoane et al., 2001) gives SMAD3 MH1 domain as aas 1-164 and SMAD4 MH1 domain as aas 1-166. These sequences do not agree with UniProt MH1 domain sequence numbering. HA-FOXO3 (N terminal tag) (PMID:10102273 Sup Ref 1, Brunet et al., 1999) is Human in origin. HA-FOXO1 (N-terminal tag) (PMID:11696581 Sup Ref 10, Nakae et al., 2001) is Murine in origin. HA-FOXO4 (tag location unknown) (PMID:10217147 Sup Ref 6, Kops et al., 1999) is Human in origin. Myc-FOXG1 (tag location unknown) PMID:10938097 Sup Ref 2, Dou et al., 2000) is Murine in origin. j-massague@ski.mskcc.org Accepted 2016-JUL-14 AT 17:06 BST AT 17:06 BST by PPORRAS Cell 2004 Only protein-protein interactions imex curation imex curation Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004 imex curation Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org 04-Aug-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) This is meant to be a "sequential IP";however "Precipitates were subjected to anti-Smad immunoblotting"-but as the antibody(s) used and their targets are not known, the bands present that are identified as Smad 3 (transfected?) and Smad 4 (endogenous) have not been annotated. No "anti-smad" antibody is listed in the methods. j-massague@ski.mskcc.org Accepted 2016-JUL-14 AT 17:06 BST AT 17:06 BST by PPORRAS Cell 2004 Only protein-protein interactions imex curation imex curation Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004 imex curation Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) j-massague@ski.mskcc.org 04-Aug-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-hacat Homo sapiens immortalised keratinocytes hacat human immortalised keratinocytes ch-ip chromatin immunoprecipitation assay ch-ip primer specific pcr primer specific pcr Seoane J., Le HV., Shen L., Anderson SA., Massagué J. IBD - Inflammatory bowel disease No species of origin, nor the exact identity of TGF-beta is stated in this publication. as the origin of TGF-beta is not known the only possible causality statement I think I could make is for Supp Fig 1A (without TGF-beta)- but I am not sure the difference in results between transfected Foxo4 and no transfection is significant -none of the other results in other figures are. (e.g. Foxo4 increases expression at RNA level of p21Cip1 gene) ChIP data using Smad2/3 was not recorded since the antibodies used were not specific enough to ascertain the protein identity. j-massague@ski.mskcc.org Accepted 2016-JUL-14 AT 17:06 BST AT 17:06 BST by PPORRAS Cell 2004 Only protein-protein interactions imex curation imex curation smad3_human Mothers against decapentaplegic homolog 3 SMAD3 MADH3 SMAD family member 3 JV15-2 protein protein human Homo sapiens Human MSSILPFTPPIVKRLLGWKKGEQNGQEEKWCEKAVKSLVKKLKKTGQLDELEKAITTQNVNTKCITIPRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELRAMELCEFAFNMKKDEVCVNPYHYQRVETPVLPPVLVPRHTEIPAEFPPLDDYSHSIPENTNFPAGIEPQSNIPETPPPGYLSEDGETSDHQMNHSMDAGSPNLSPNPMSPAHNNLDLQPVTYCEPAFWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS DIP protein P84022 original sequence version: 57 DIP protein P84022 original sequence version: 112 DIP protein P84022-1 original sequence version: 146 DIP protein P84022-1 original sequence version: 111 46DF5E8B371321AC cdkn1a_human_gene Human CDKN1A gene gene gene human Homo sapiens Human foxh1_mouse Forkhead box protein H1 Foxh1 Fast1 Fast2 Forkhead activin signal transducer 1 Forkhead activin signal transducer 2 protein protein mouse Mus musculus Mouse MASGWDLASTYTPTTPSPQLALAPAQGYLPCMGPRDNSQLRPPEAESLSKTPKRRKKRYLRHDKPPYTYLAMIALVIQAAPFRRLKLAQIIRQVQAVFPFFRDDYEGWKDSIRHNLSSNRCFHKVPKDPAKPQAKGNFWAVDVSLIPAEALRLQNTALCRRWQNRGTHRAFAKDLSPYVLHGQPYQPPSPPPPPREGFSIKSLLGDPGKESTWPQHPGLPGQSTAAQAGTLSKGEEGMGTGPSSSSETPLWPLCSLPGPTIIEGESSQGEVIRPSPVTPDQGSWPLHLLEDSADSRGVPRRGSRASLWGQLPTSYLPIYTPNVVMPLATLPTTSCPQCPSSASPAYWSVGTESQGSQDLLCDLDSLFQGVPPNKSIYDVWVSHPRDLAAPAPGWLLSWYSM Transcriptional activator. Recognizes and binds to the DNA sequence 5'-TGT[GT][GT]ATT-3'. Required for induction of the goosecoid (GSC) promoter by TGF-beta or activin signaling. Forms a transcriptionally active complex containing FOXH1/SMAD2/SMAD4 on a site on the GSC promoter called TARE (TGF-beta/activin response element) mint FB11DC9779DF1454 foxo1_mouse Forkhead box protein O1 Foxo1 Fkhr Foxo1a Forkhead box protein O1A Forkhead in rhabdomyosarcoma protein protein mouse Mus musculus Mouse MAEAPQVVETDPDFEPLPRQRSCTWPLPRPEFNQSNSTTSSPAPSGGAAANPDAAASLASASAVSTDFMSNLSLLEESEDFARAPGCVAVAAAAAASRGLCGDFQGPEAGCVHPAPPQPPPTGPLSQPPPVPPSAAAAAGPLAGQPRKTSSSRRNAWGNLSYADLITKAIESSAEKRLTLSQIYEWMVKSVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIRVQNEGTGKSSWWMLNPEGGKSGKSPRRRAASMDNNSKFAKSRGRAAKKKASLQSGQEGPGDSPGSQFSKWPASPGSHSNDDFDNWSTFRPRTSSNASTISGRLSPIMTEQDDLGDGDVHSLVYPPSAAKMASTLPSLSEISNPENMENLLDNLNLLSSPTSLTVSTQSSPGSMMQQTPCYSFAPPNTSLNSPSPNYSKYTYGQSSMSPLPQMPMQTLQDSKSSYGGLNQYNCAPGLLKELLTSDSPPHNDIMSPVDPGVAQPNSRVLGQNVMMGPNSVMPAYGSQASHNKMMNPSSHTHPGHAQQTASVNGRTLPHVVNTMPHTSAMNRLTPVKTPLQVPLSHPMQMSALGSYSSVSSCNGYGRMGVLHQEKLPSDLDGMFIERLDCDMESIIRNDLMDGDTLDFNFDNVLPNQSFPHSVKTTTHSWVSG 3FF58636EA85205F foxo1_human Forkhead box protein O1 FOXO1 FKHR FOXO1A Forkhead box protein O1A Forkhead in rhabdomyosarcoma protein protein human Homo sapiens Human MAEAPQVVEIDPDFEPLPRPRSCTWPLPRPEFSQSNSATSSPAPSGSAAANPDAAAGLPSASAAAVSADFMSNLSLLEESEDFPQAPGSVAAAVAAAAAAAATGGLCGDFQGPEAGCLHPAPPQPPPPGPLSQHPPVPPAAAGPLAGQPRKSSSSRRNAWGNLSYADLITKAIESSAEKRLTLSQIYEWMVKSVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIRVQNEGTGKSSWWMLNPEGGKSGKSPRRRAASMDNNSKFAKSRSRAAKKKASLQSGQEGAGDSPGSQFSKWPASPGSHSNDDFDNWSTFRPRTSSNASTISGRLSPIMTEQDDLGEGDVHSMVYPPSAAKMASTLPSLSEISNPENMENLLDNLNLLSSPTSLTVSTQSSPGTMMQQTPCYSFAPPNTSLNSPSPNYQKYTYGQSSMSPLPQMPIQTLQDNKSSYGGMSQYNCAPGLLKELLTSDSPPHNDIMTPVDPGVAQPNSRVLGQNVMMGPNSVMSTYGSQASHNKMMNPSSHTHPGHAQQTSAVNGRPLPHTVSTMPHTSGMNRLTQVKTPVQVPLPHPMQMSALGGYSSVSSCNGYGRMGLLHQEKLPSDLDGMFIERLDCDMESIIRNDLMDGDTLDFNFDNVLPNQSFPHSVKTTTHSWVSG DIP protein Q12778 original sequence version: 132 DIP protein Q12778 original sequence version: 84 6DEF6C994BDFDBAB foxo3_human Forkhead box protein O3 FOXO3 FKHRL1 FOXO3A Forkhead in rhabdomyosarcoma-like 1 AF6q21 protein protein protein human Homo sapiens Human MAEAPASPAPLSPLEVELDPEFEPQSRPRSCTWPLQRPELQASPAKPSGETAADSMIPEEEDDEDDEDGGGRAGSAMAIGGGGGSGTLGSGLLLEDSARVLAPGGQDPGSGPATAAGGLSGGTQALLQPQQPLPPPQPGAAGGSGQPRKCSSRRNAWGNLSYADLITRAIESSPDKRLTLSQIYEWMVRCVPYFKDKGDSNSSAGWKNSIRHNLSLHSRFMRVQNEGTGKSSWWIINPDGGKSGKAPRRRAVSMDNSNKYTKSRGRAAKKKAALQTAPESADDSPSQLSKWPGSPTSRSSDELDAWTDFRSRTNSNASTVSGRLSPIMASTELDEVQDDDAPLSPMLYSSSASLSPSVSKPCTVELPRLTDMAGTMNLNDGLTENLMDDLLDNITLPPSQPSPTGGLMQRSSSFPYTTKGSGLGSPTSSFNSTVFGPSSLNSLRQSPMQTIQENKPATFSSMSHYGNQTLQDLLTSDSLSHSDVMMTQSDPLMSQASTAVSAQNSRRNVMLRNDPMMSFAAQPNQGSLVNQNLLHHQHQTQGALGGSRALSNSVSNMGLSESSSLGSAKHQQQSPVSQSMQTLSDSLSGSSLYSTSANLPVMGHEKFPSDLDLDMFNGSLECDMESIIRSELMDADGLDFNFDSLISTQNVVGLNVGNFTGAKQASSQSWVPG E5B4E830665A9982 DIP protein O43524 original sequence version: 82 E5B4E830665A9982 myc_human Myc proto-oncogene protein BHLHE39 Transcription factor p64 MYC Proto-oncogene c-Myc Class E basic helix-loop-helix protein 39 protein protein human Homo sapiens Human MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA DIP protein P01106 original sequence version: 195 DIP protein P01106 original sequence version: 196 DIP protein P01106 original sequence version: 148 DIP protein P01106-1 original sequence version: 161 DIP protein P01106 original sequence version: 182 DIP protein P01106 original sequence version: 197 DIP protein P01106-1 original sequence version: 208 DIP protein P01106 original sequence version: 121 DIP protein P01106-1 original sequence version: 144 DIP protein P01106 original sequence version: 144 ED5C028029A4C5D1 foxo4_human Forkhead box protein O4 FOXO4 AFX AFX1 MLLT7 Fork head domain transcription factor AFX1 protein protein human Homo sapiens Human MDPGNENSATEAAAIIDLDPDFEPQSRPRSCTWPLPRPEIANQPSEPPEVEPDLGEKVHTEGRSEPILLPSRLPEPAGGPQPGILGAVTGPRKGGSRRNAWGNQSYAELISQAIESAPEKRLTLAQIYEWMVRTVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIKVHNEATGKSSWWMLNPEGGKSGKAPRRRAASMDSSSKLLRGRSKAPKKKPSVLPAPPEGATPTSPVGHFAKWSGSPCSRNREEADMWTTFRPRSSSNASSVSTRLSPLRPESEVLAEEIPASVSSYAGGVPPTLNEGLELLDGLNLTSSHSLLSRSGLSGFSLQHPGVTGPLHTYSSSLFSPAEGPLSAGEGCFSSSQALEALLTSDTPPPPADVLMTQVDPILSQAPTLLLLGGLPSSSKLATGVGLCPKPLEAPGPSSLVPTLSMIAPPPVMASAPIPKALGTPVLTPPTEAASQDRMPQDLDLDMYMENLECDMDNIISDLMDEGEGLDFNFEPDP 0C71C8E2167CEE68 smad2_human Mothers against decapentaplegic homolog 2 SMAD2 MADH2 MADR2 Mad-related protein 2 SMAD family member 2 JV18-1 protein protein human Homo sapiens Human MSSILPFTPPVVKRLLGWKKSAGGSGGAGGGEQNGQEEKWCEKAVKSLVKKLKKTGRLDELEKAITTQNCNTKCVTIPSTCSEIWGLSTPNTIDQWDTTGLYSFSEQTRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEVCVNPYHYQRVETPVLPPVLVPRHTEILTELPPLDDYTHSIPENTNFPAGIEPQSNYIPETPPPGYISEDGETSDQQLNQSMDTGSPAELSPTTLSPVNHSLDLQPVTYSEPAFWCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSVRCSSMS DIP protein Q15796 original sequence version: 174 DIP protein Q15796 original sequence version: 100 DIP protein Q15796-1 original sequence version: 141 95406DB5FC0AA4C9 p21_cip1_oligo_sbr_distal_promoter_region SBR ds dna double stranded deoxyribonucleic acid ds DNA ds dna human Homo sapiens Human CCCAAAGTAAACAGACAGACAATGTCTAGTCTATTT foxg1_mouse Forkhead box protein G1 Foxg1 Fkhl1 Foxg1b Hfhbf1 Brain factor 1 Forkhead-related protein FKHL1 protein protein mouse Mus musculus Mouse MLDMGDRKEVKMIPKSSFSINSLVPEAVQNDNHHASHGHHNSHHPQHHHHHHHHHHPPPPAPQPPPPPPQQQQQQPPPAPQPPQARGAPAADDDKGPQPLLLPPSTALDGAKADALGAKGEPGGGPAELAPVGPDEKEKGAGAGGEEKKGAGEGGKDGEGGKEGDKKNGKYEKPPFSYNALIMMAIRQSPEKRLTLNGIYEFIMKNFPYYRENKQGWQNSIRHNLSLNKCFVKVPRHYDDPGKGNYWMLDPSSDDVFIGGTTGKLRRRSTTSRAKLAFKRGARLTSTGLTFMDRAGSLYWPMSPFLSLHHPRASSTLSYNGTTSAYPSHPMPYSSVLTQNSLGNNHSFSTANGLSVDRLVNGEIPYATHHLTAAALAASVPCGLSVPCSGTYSLNPCSVNLLAGQTSYFFPHVPHPSMTSQTSTSMSARAASSSTSPQAPSTLPCESLRPSLPSFTTGLSGGLSDYFTHQNQGSSSNPLIH FF6786C3513AB452 smad4_human Mothers against decapentaplegic homolog 4 SMAD4 DPC4 MADH4 SMAD family member 4 Deletion target in pancreatic carcinoma 4 protein protein human Homo sapiens Human MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITAITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFDLKCDSVCVNPYHYERVVSPGIDLSGLTLQSNAPSSMMVKDEYVHDFEGQPSLSTEGHSIQTIQHPPSNRASTETYSTPALLAPSESNATSTANFPNIPVASTSQPASILGGSHSEGLLQIASGPQPGQQQNGFTGQPATYHHNSTTTWTGSRTAPYTPNLPHHQNGHLQHHPPMPPHPGHYWPVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD DIP protein Q13485 original sequence version: 149 DIP protein Q13485 original sequence version: 177 7EE3C4647712DA90 myc-cdkn1a_human_gene-2 7 14 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey proximal_promoter_region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 1B TGF-beta smad4-cdkn1a_human_gene-1 7 19 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey distal_promoter_region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 1B-2, 2D TGF-beta In the presence of TGF-beta, a band was seen when using a anti-SMAD 2+3 antibody. p21_cip1_oligo_sbr_distal_promoter_region-1 3 19 unspecified role unspecified role prey prey human-hacat Homo sapiens immortalised keratinocytes hacat human immortalised keratinocytes 17 unspecified role unspecified role bait bait region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined A15G,C16G,G18T,A19C mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain AC GG certain certain sequence position certain certain certain sequence position certain GA TC chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association 1 C/F TGF-beta;No binding is seen in the absence of TGF-beta c-Myc did not diminish Smad binding to the isolated SBR foxo3-cdkn1a_human_gene-1 7 13 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey distal_promoter_region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 2D TGF-beta In the presence of TGF-beta, a band was seen when using a anti-SMAD 2+3 antibody. smad4-p21_cip1_oligo_sbr_distal_promoter_region-1 2 17 unspecified role unspecified role prey prey A15G,C16G,G18T,A19C mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain AC GG certain certain sequence position certain certain certain sequence position certain GA TC region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined delta_sbe_3,4 required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 19 unspecified role unspecified role bait bait human-hacat Homo sapiens immortalised keratinocytes hacat human immortalised keratinocytes association association 2E TGF-beta please check the Delta 3/4SBe construct looks as it should be, as the arrow for "3" under Figure 1A does not correspond to where the deletion is shown (I think) on top of the image. foxo3-smad4-2 4 13 unspecified role unspecified role bait bait 19 unspecified role unspecified role prey prey physical association physical association 3A TGF-beta foxo1-smad4-1 4 12 unspecified role unspecified role bait bait 19 unspecified role unspecified role prey prey physical association physical association 3Ai TGF-beta foxo1-smad4-2 4 15 unspecified role unspecified role bait bait 19 unspecified role unspecified role prey prey physical association physical association 3Aii TGF-beta smad3-foxo3-1 1 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association 3C smad3-foxo1-1 1 11 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 3C-2 foxo4-foxg1-1 1 18 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 15 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 5E-3 smad4-foxo3-2 1 19 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association 3C-4 smad4-foxo1-1 1 11 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 19 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 3C-5 smad4-foxo4-1 1 19 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 15 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association 3C-6 smad3-foxo1-2 1 11 unspecified role unspecified role bait bait n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 18 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 5E smad3-foxo3-2 1 18 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role bait bait n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association 5E-2, 5G In fig 5G endogenous Smad 4 and 3 are stated to have been detected. Fig 5G:I dont see how they show Smad3 and 4 on the same blot; the antibody for 2/3 detects both smads- so cannot say which smad it is, as M.Wts of SMad 2 and 3 are very similar;Smad 4 is higher (60 kDa as oppossed to 52/48). Birgit thinks they may have mixed antibodies? foxo3-p21_cip1_oligo_sbr_distal_promoter_region-1 2 17 unspecified role unspecified role prey prey A5G,A6G,G7A,A9C,A10C mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain AAG GGA certain certain sequence position certain certain certain sequence position certain AA CC region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 13 unspecified role unspecified role bait bait possible_HA_or_FLAG_or_HIS_or_GST tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 association association Supp 1B foxo4-p21_cip1_oligo_sbr_distal_promoter_region-1 2 17 unspecified role unspecified role prey prey A5G,A6G,G7A,A9C,A10C mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain AAG GGA certain certain sequence position certain certain certain sequence position certain AA CC region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 15 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 association association Supp 1B-2 smad3-foxo3-3 5 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined mh1_domain sufficient to bind sufficient binding region sufficient to bind range range range range 13 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Supp 2A smad3-foxo3-4 5 19 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined mh1_domain sufficient to bind sufficient binding region sufficient to bind range range range range 13 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Supp 2Ai smad2-foxh1-3 1 10 unspecified role unspecified role prey prey n_terminus myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 16 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined MH2_domain sufficient to bind sufficient binding region sufficient to bind range range range range physical association physical association Supp 2A smad3-foxh1-1 1 10 unspecified role unspecified role prey prey n_terminus myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined MH2_domain sufficient to bind sufficient binding region sufficient to bind range range range range physical association physical association Supp 2A-2 foxo3-smad4-4 5 19 unspecified role unspecified role prey prey possible_ flag_or_gst tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n_terminus sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Supp 2B foxo3-smad4-5 5 8 unspecified role unspecified role prey prey possible_ flag_or_gst tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n_terminus sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Supp 2Bi smad3-foxo3-5 3 8 unspecified role unspecified role bait bait n_terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range mh1_domain_+_linker sufficient to bind sufficient binding region sufficient to bind range range range range ecoli Escherichia coli (strain K12) 13 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey n_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction Supp 2D smad4-foxo3-3 3 19 unspecified role unspecified role bait bait n_terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range mh1_domain-+-linker sufficient to bind sufficient binding region sufficient to bind range range range range ecoli Escherichia coli (strain K12) 13 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey n_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction Supp 2D foxo3-foxg1-1 1 18 unspecified role unspecified role bait bait n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range c_terminal_region required to bind necessary binding region required to bind range range certain certain sequence position certain 13 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Supp 3B foxg1-foxo3-1 1 18 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined partial_ct1_region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain n_terminal_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Supp 3C A construct containing amino acids 444–674 does not bind FoxG1. foxo3-foxg1-2 3 18 unspecified role unspecified role prey prey n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 13 unspecified role unspecified role bait bait n_terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Supp 3C The main text states that:- "the interacting domains were mapped to the N-terminal half of FoxO3 and the C-terminal region of FoxG1". Can you work out which band refers to which of the constructs? - I would read the result as the full length and (possible) NT2 domain (residues 139–280) are interacting, but it is hard to work out which band each part of the text is referring to.. foxo3-foxg1-3 6 18 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Supp 3E smad3-foxo3-6 1 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 15 unspecified role unspecified role prey prey n_terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association 3C-2