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/ A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell (1535-6108) 2008 imex curation Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions 16 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org Accepted 2008-Not a month!-15 by DAVET. 09-April-2009: Contacted by JYOTI. Cancer Cell (1535-6108) 2008 davet Only protein-protein interactions 16 imex curation A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell (1535-6108) 2008 imex curation Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions 16 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org Accepted 2008-Not a month!-15 by DAVET. 09-April-2009: Contacted by JYOTI. Cancer Cell (1535-6108) 2008 davet Only protein-protein interactions 16 imex curation A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell (1535-6108) 2008 imex curation Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions 16 human-dohh2 Human DOHH2 lymphoma follicular B cell dohh2 DoHH2 lymphoma, follicular, B cells crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org Accepted 2008-Not a month!-15 by DAVET. 09-April-2009: Contacted by JYOTI. Cancer Cell (1535-6108) 2008 davet Only protein-protein interactions 16 imex curation A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell (1535-6108) 2008 imex curation Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions 16 in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps predetermined predetermined participant predetermined Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org Accepted 2008-Not a month!-15 by DAVET. 09-April-2009: Contacted by JYOTI. Cancer Cell (1535-6108) 2008 davet Only protein-protein interactions 16 imex curation A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell (1535-6108) 2008 imex curation Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions 16 in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org Accepted 2008-Not a month!-15 by DAVET. 09-April-2009: Contacted by JYOTI. Cancer Cell (1535-6108) 2008 davet Only protein-protein interactions 16 imex curation A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell (1535-6108) 2008 imex curation Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org 09-April-2009: Contacted by JYOTI. davet Only protein-protein interactions 16 human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Kolluri SK., Zhu X., Zhou X., Lin B., Chen Y., Sun K., Tian X., Town J., Cao X., Lin F., Zhai D., Kitada S., Luciano F., O'Donnell E., Cao Y., He F., Lin J., Reed JC., Satterthwait AC., Zhang XK. Apoptosis - Interactions involving proteins with a function related to apoptosis Bismaleimidohexane (BMH) cross-linker. Additional information on source of interacting proteins supplied by the authors asat@burnham.org, xzhang@burnham.org Accepted 2008-Not a month!-15 by DAVET. 09-April-2009: Contacted by JYOTI. Cancer Cell (1535-6108) 2008 davet Only protein-protein interactions 16 imex curation q07817-1 Bcl-2-like protein 1 BCLX Apoptosis regulator Bcl-X BCL2L1 BCL2L Bcl-xL protein protein human Homo sapiens Human MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK DIP protein Q07817 original sequence version: 82 DIP protein Q07817 original sequence version: 86 DIP protein Q07817-1 original sequence version: 100 DIP protein Q07817-1 original sequence version: 108 DIP protein Q07817 original sequence version: 131 DIP protein Q07817-1 original sequence version: 110 DIP protein Q07817-1 original sequence version: 135 DIP protein Q07817-1 original sequence version: 131 DIP protein Q07817-1 original sequence version: 172 DIP protein Q07817-1 original sequence version: 174 DIP protein Q07817 original sequence version: 174 DIP protein Q07817-1 original sequence version: 189 DIP protein Q07817-1 original sequence version: 192 DIP protein Q07817 original sequence version: 190 E09D3CDD851AE9BE b2l10_human Bcl-2-like protein 10 BCL2L10 BCLB Apoptosis regulator Bcl-B Anti-apoptotic protein NrH protein protein human Homo sapiens Human MVDQLRERTTMADPLRERTELLLADYLGYCAREPGTPEPAPSTPEAAVLRSAAARLRQIHRSFFSAYLGYPGNRFELVALMADSVLSDSPGPTWGRVVTLVTFAGTLLERGPLVTARWKKWGFQPRLKEQEGDVARDCQRLVALLSSRLMGQHRAWLQAQGGWDGFCHFFRTPFPLAFWRKQLVQAFLSCLLTTAFIYLWTRLL 4B46B2DC472475CB b2la1_human Bcl-2-related protein A1 Protein BFL-1 Hemopoietic-specific early response protein Protein GRS BCL2A1 BCL2L5 BFL1 GRS HBPA1 Bcl-2-like protein 5 protein protein human Homo sapiens Human MTDCEFGYIYRLAQDYLQCVLQIPQPGSGPSKTSRVLQNVAFSVQKEVEKNLKSCLDNVNVVSVDTARTLFNQVMEKEFEDGIINWGRIVTIFAFEGILIKKLLRQQIAPDVDTYKEISYFVAEFIMNNTGEWIRQNGGWENGFVKKFEPKSGWMTFLEVTGKICEMLSLLKQYC 329D98AF2BE07A0D bcl2_human Apoptosis regulator Bcl-2 BCL2 protein protein human Homo sapiens Human MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK DIP protein P10415 original sequence version: 148 DIP protein P10415 original sequence version: 125 DIP protein P10415 original sequence version: 151 DIP protein P10415 original sequence version: 204 DIP protein P10415 original sequence version: 149 3C49F2B714DC9CCB nr4a1_human Nuclear receptor subfamily 4 group A member 1 Orphan nuclear receptor HMR Early response protein NAK1 Orphan nuclear receptor TR3 ST-59 GFRP1 HMR NAK1 NR4A1 Nuclear hormone receptor NUR/77 Testicular receptor 3 protein protein human Homo sapiens Human MPCIQAQYGTPAPSPGPRDHLASDPLTPEFIKPTMDLASPEAAPAAPTALPSFSTFMDGYTGEFDTFLYQLPGTVQPCSSASSSASSTSSSSATSPASASFKFEDFQVYGCYPGPLSGPVDEALSSSGSDYYGSPCSAPSPSTPSFQPPQLSPWDGSFGHFSPSQTYEGLRAWTEQLPKASGPPQPPAFFSFSPPTGPSPSLAQSPLKLFPSQATHQLGEGESYSMPTAFPGLAPTSPHLEGSGILDTPVTSTKARSGAPGGSEGRCAVCGDNASCQHYGVRTCEGCKGFFKRTVQKNAKYICLANKDCPVDKRRRNRCQFCRFQKCLAVGMVKEVVRTDSLKGRRGRLPSKPKQPPDASPANLLTSLVRAHLDSGPSTAKLDYSKFQELVLPHFGKEDAGDVQQFYDLLSGSLEVIRKWAEKIPGFAELSPADQDLLLESAFLELFILRLAYRSKPGEGKLIFCSGLVLHRLQCARGFGDWIDSILAFSRSLHSLLVDVPAFACLSALVLITDRHGLQEPRRVEELQNRIASCLKEHVAAVAGEPQPASCLSRLLGKLPELRTLCTQGLQRIFYLKLEDLVPPPPIIDKIFMDTLPF DIP protein P22736 original sequence version: 156 41DAAEA7C25FDA22 bax_human Apoptosis regulator BAX BAX BCL2L4 Bcl-2-like protein 4 protein protein human Homo sapiens Human MDGSGEQPRGGGPTSSEQIMKTGALLLQGFIQDRAGRMGGEAPELALDPVPQDASTKKLSECLKRIGDELDSNMELQRMIAAVDTDSPREVFFRVAADMFSDGNFNWGRVVALFYFASKLVLKALCTKVPELIRTIMGWTLDFLRERLLGWIQDQGGWDGLLSYFGTPTWQTVTIFVAGVLTASLTIWKKMG DIP protein Q07812 original sequence version: 73 DIP protein Q07812 original sequence version: 156 DIP protein Q07812 original sequence version: 92 DIP protein Q07812 original sequence version: 94 DIP protein Q07812-1 original sequence version: 93 DIP protein Q07812 original sequence version: 95 DIP protein Q07812 original sequence version: 120 DIP protein Q07812 original sequence version: 126 DIP protein Q07812 original sequence version: 143 DIP protein Q07812 original sequence version: 172 DIP protein Q07812 original sequence version: 175 6C0CDB0A7DEE4994 nr4a1-bcl2-1 1 11 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role bait bait physical association physical association Fig. 3A HEK293T cells transfected with GFP-Nur77/489-497 or GFP-Nur77/478-504 (2 μg) together with or without Bcl-2 expression plasmid (0.8 μg) were analyzed by coimmunoprecipitation (CoIP) using anti-Bcl-2 antibody, followed by western blotting (WB) using either anti-GFP or anti-Bcl-2 antibody P10415; P22736 transfection nr4a1-bcl2-2 1 11 competitor competitor competitor prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region D-arg octomer tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain association association Fig. 3B Nur77 lacking its DNA-binding domain (DBD), Nur77/ΔDBD, bound Bcl-2 (Lin et al., 2004 B. Lin, S.K. Kolluri, F. Lin, W. Liu, Y.H. Han, X. Cao, M.I. Dawson, J.C. Reed and X.K. Zhang, Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3, Cell 116 (2004), pp. 527�540. Article | PDF (976 K) | View Record in Scopus | Cited By in Scopus (157)Lin et al., 2004), and binding was inhibited by NuBCP-9 or D-NuBCP-9 (Figure 3B). P10415; P22736 transfection Replacing L-amino acids with D-amino acids did not diminish the effect of P22736 . nr4a1-bcl2-3 2 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P10415:p.Thr69_Ser70delinsGluGlu mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain TS EE Thu Jul 07 12:16:58 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was thr69glu_ser70glu Fri Jul 22 11:05:52 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'thr69glu,ser70glu' Wed May 02 20:14:15 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr_ser69-70glu_glu' Tue Jun 30 16:45:23 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr69_Ser70delinsGluGlu' undefined mutation mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing insertion analysis insertion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Wed Jul 06 16:17:40 BST 2016 Sequence change details about this feature cannot be ascertained or do not fit with the current version of the referenced protein, so they have been deleted as a result of our quality control procedures. The original label was 'vartsplqtp' 11 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 29 34 physical association physical association Figs. 3C, 5A, 5G Interaction of NuBCP with antiapoptotic Bcl-2 family members, Bcl-B, Bfl-1, and Bcl-2. P10415; P22736 transfection Exact insertion point of the insertion mutant into the BCL-2 interacting region is not given in the text. nr4a1-bcl2l10-1 2 11 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role bait bait 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 Fig. 3C Interaction of NuBCP with antiapoptotic Bcl-2 family members, Bcl-B, Bfl-1, and Bcl-2. Q9HD36; P22736 transfection nr4a1-bcl2la1-1 2 9 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 11 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig. 3C Interaction of NuBCP with antiapoptotic Bcl-2 family members, Bcl-B, Bfl-1, and Bcl-2. Q16548; P22736 transfection nr4a1-bcl2-4 4 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey region fitc labelled fluorescein isothiocyanate label fluorescein isothiocyanate labbeled FITC labelled FITC labelled fluorescein isothiocyanate labbeled fitc labelled undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P22736:p.[Phe489Ala;Leu497Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain F A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 19:24:11 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe489ala,leu497ala' Tue Jun 30 17:49:19 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Phe489Ala;Leu497Ala]' chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association Figs. 3D, 4E, 5B, 5H We next used fluorescence polarization (FP) assays to determine whether NuBCP-9s bind Bcl-2. Replacing L-amino acids of P22736 with D-amino acids did not diminish its effect. pH=7.6, Temp=25C nr4a1-bcl2-5 4 11 competitor competitor competitor prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P22736:p.[Phe489Ala;Leu497Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain F A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 18:56:28 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe489ala,leu497ala' Tue Jun 30 16:41:19 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Phe489Ala;Leu497Ala]' chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey region fitc labelled fluorescein isothiocyanate label fluorescein isothiocyanate labbeled FITC labelled FITC labelled fluorescein isothiocyanate labbeled fitc labelled undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) association association Figs. 3E, 4F, 5B, 5H We next used fluorescence polarization (FP) assays to determine whether NuBCP-9s bind Bcl-2. Replacing L-amino acids of P22736 with D-amino acids did not diminish the effect of P22736. Kd of D-enantiomer of P22736 = 1.51E-7M EC50 inhibition by D-enantiomer of P22736 = 4.5E-6M, Ki=1.9E-6M EC50 inhibition by P22736 = 3.2E-6M, Ki=1.2E-6M pH=7.6, Temp=20C nr4a1-bcl2-6 5 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P22736:p.[Phe489Ala;Leu497Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 15:42:50 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe489ala,leu497ala' Tue Jun 30 20:24:39 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Phe489Ala;Leu497Ala]' chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction Fig. 4C, 4d, 4E, 5C, 5D, Supp. Fig. 8 We next determined whether NuBCPs can induce a conformational change of purified GST-Bcl-2 protein using circular dichroism (CD) analysis. Replacing L-amino acids of P22736 with D-amino acids did not diminish its effect. pH=7.6, Temp=25C Binding of D-enantiomer of P22736 Kd=2.5E-6M nr4a1-bcl2-7 5 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction Fig. 5I CD spectra for the binding of NuBCPs (30 μM) to GST-Bcl-2/29�90 (2 μM). bax-1 6 12 unspecified role unspecified role neutral component neutral component physical association physical association Homodimerization identified by a calibrated change in molecular weight of the protein. Fig. 6A, 6B NR4A1 (P22736), amino acids 489-497. Replacing the L-amino acids with D-amino acids did not diminish its effect. In vitro assays using isolated mitochondria showed that both NuBCP-9 and D-NuBCP-9 induced Bax dimerization, trimerization, and especially oligomerization (Figure 6A) in a concentration-dependent manner Stimulation by NR4A1 required the presence of BCL2. bax-2 6 12 unspecified role unspecified role neutral component neutral component physical association physical association Homotrimerization identified by a calibrated change in molecular weight of the protein. Fig. 6A, 6B NR4A1 (P22736), amino acids 489-497. Replacing the L-amino acids with D-amino acids did not diminish its effect. In vitro assays using isolated mitochondria showed that both NuBCP-9 and D-NuBCP-9 induced Bax dimerization, trimerization, and especially oligomerization (Figure 6A) in a concentration-dependent manner (Figure 6B) Stimulation by NR4A1 required the presence of BCL2. bax-3 3 12 unspecified role unspecified role neutral component neutral component physical association physical association Homotrimerization identified by a calibrated change in molecular weight of the protein. Fig. 6C NR4A1 (P22736), amino acids 489-497. Replacing the L-amino acids with D-amino acids did not diminish its effect. Bax dimerization/oligomerization in DoHH2 cells. DoHH2 cells express high levels of BCL2. bax-1 3 12 unspecified role unspecified role neutral component neutral component physical association physical association Homodimerization identified by a calibrated change in molecular weight of the protein. Fig. 6C NR4A1 (P22736), amino acids 489-497. Replacing the L-amino acids with D-amino acids did not diminish its effect. Bax dimerization/oligomerization in DoHH2 cells. DoHH2 cells express high levels of BCL2. bcl2-1 2 10 unspecified role unspecified role bait bait region delta bh4 sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range range range certain certain sequence position certain region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey bh4 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association GFP-BH4 or Bcl-2/ΔBH4 was transfected alone or together into HEK293T cells, and their interaction was analyzed by CoIP. Fig. 7A The BH4 domain could not bind full-length Bcl-2, likely due to inaccessibility of a BH4-binding site in the C-terminal region Coordinates of the deletion mutants not given by the author so have been inferred from the UniProtKB entry. bcl2-2 2 10 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey bh4 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association . Similarly, addition of NuBCP-9 and its enantiomer induced the binding of the BH4 domain to Bcl-2 (Figure 7C). Figs. 7B, 7C NR4A1 - 489-497. Interaction of BH4 domain with full length protein only occurs in the presence of this agonist, suggesting that binding of NR4A1 with Bcl-2 reorganizes Bcl-2, leading to the exposure of the BH4-binding site in the C-terminal region. bcl2-bclx-1 4 7 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component bh3 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region F5M fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction FP assay of binding of Bcl-2 BH3 peptide to Bcl-XL. pH = 7.4, Temp. = 25C Fig. 8D