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/ 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. Cancer Cell (1535-6108) 2009 imex curation Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Details of constructs supplied by authors. dyu@mdanderson.org 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 5 human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Details of constructs supplied by authors. dyu@mdanderson.org Accepted 2010-APR-25 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 5 imex curation 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. Cancer Cell (1535-6108) 2009 imex curation Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Details of constructs supplied by authors. dyu@mdanderson.org 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 5 human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line ch-ip chromatin immunoprecipitation assay ch-ip nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Details of constructs supplied by authors. dyu@mdanderson.org Accepted 2010-APR-25 by ORCHARD. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 5 imex curation 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. Cancer Cell (1535-6108) 2009 imex curation Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Details of constructs supplied by authors. dyu@mdanderson.org 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 5 human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line pull down pull down western blot western blot Immuno blot Immuno blot Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Details of constructs supplied by authors. dyu@mdanderson.org Accepted 2010-APR-25 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 5 imex curation 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. 14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. Cancer Cell (1535-6108) 2009 imex curation Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Details of constructs supplied by authors. dyu@mdanderson.org 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 5 human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Lu J., Guo H., Treekitkarnmongkol W., Li P., Zhang J., Shi B., Ling C., Zhou X., Chen T., Chiao PJ., Feng X., Seewaldt VL., Muller WJ., Sahin A., Hung MC., Yu D. Apoptosis - Interactions involving proteins with a function related to apoptosis Details of constructs supplied by authors. dyu@mdanderson.org Accepted 2010-APR-25 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 5 imex curation 1433z_human 14-3-3 protein zeta/delta YWHAZ Protein kinase C inhibitor protein 1 protein protein human Homo sapiens Human MDKNELVQKAKLAEQAERYDDMAACMKSVTEQGAELSNEERNLLSVAYKNVVGARRSSWRVVSSIEQKTEGAEKKQQMAREYREKIETELRDICNDVLSLLEKFLIPNASQAESKVFYLKMKGDYYRYLAEVAAGDDKKGIVDQSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFYYEILNSPEKACSLAKTAFDEAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDTQGDEAEAGEGGEN DIP protein P63104 original sequence version: 144 D464DF2286BBFE60 rac1_human Ras-related C3 botulinum toxin substrate 1 RAC1 MIG5 p21-Rac1 Ras-like protein TC25 Cell migration-inducing gene 5 protein TC25 protein protein human Homo sapiens Human MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHHCPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRGLKTVFDEAIRAVLCPPPVKKRKRKCLLL ACEDF83A45E5EA67 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 tgfr1_human TGF-beta receptor type-1 TGFBR1 Transforming growth factor-beta receptor type I TGF-beta type I receptor Serine/threonine-protein kinase receptor R4 Activin receptor-like kinase 5 ALK5 SKR4 Activin A receptor type II-like protein kinase of 53kD protein protein human Homo sapiens Human MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCFVCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLSQLSQQEGIKM DIP protein P36897 original sequence version: 161 179F11404725DDCB zeb2_human_gene Human ZFHX1B gene, promoter gene gene human Homo sapiens Human pak1_human Serine/threonine-protein kinase PAK 1 PAK1 p21-activated kinase 1 p65-PAK Alpha-PAK protein protein human Homo sapiens Human MSNNGLDIQDKPPAPPMRNTSTMIGAGSKDAGTLNHGSKPLPPNPEEKKKKDRFYRSILPGDKTNKKKEKERPEISLPSDFEHTIHVGFDAVTGEFTGMPEQWARLLQTSNITKSEQKKNPQAVLDVLEFYNSKKTSNSQKYMSFTDKSAEDYNSSNALNVKAVSETPAVPPVSEDEDDDDDDATPPPVIAPRPEHTKSVYTRSVIEPLPVTPTRDVATSPISPTENNTTPPDALTRNTEKQKKKPKMSDEEILEKLRSIVSVGDPKKKYTRFEKIGQGASGTVYTAMDVATGQEVAIKQMNLQQQPKKELIINEILVMRENKNPNIVNYLDSYLVGDELWVVMEYLAGGSLTDVVTETCMDEGQIAAVCRECLQALEFLHSNQVIHRDIKSDNILLGMDGSVKLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRKAYGPKVDIWSLGIMAIEMIEGEPPYLNENPLRALYLIATNGTPELQNPEKLSAIFRDFLNRCLEMDVEKRGSAKELLQHQFLKIAKPLSSLTPLIAAAKEATKNNH DIP protein Q13153 original sequence version: 161 DIP protein Q13153 original sequence version: 122 DIP protein Q13153 original sequence version: 134 DIP protein Q13153 original sequence version: 165 1A95CD5F2195CD7B smad3-zfhx1b 2 9 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait association association 14-3-3-zeta-mediated TGF-beta/Smads activation contributed to ZFHX1B transcriptional upregulation. smad3 binding to the ZFHX1B promoter was observed in MCF10A cells over expressing 14-3-3-zeta alone, and also when co-expressing ErbB2. Fig 4C. 1433z-tgfr1 4 8 unspecified role unspecified role prey prey 5 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 Fig 4G. 14-3-3z and TGF-beta receptor type-1 coexisted in the same complex. 1433z-tgfr1-1 3 8 unspecified role unspecified role prey prey region from Kinase domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 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 physical association physical association Fig 4I. 14-3-3z and TGF-beta receptor type-1 coexisted in the same complex and the binding region is between amino acid 210 and 370 in the kinase domain of TGF-beta receptor type-1. 1433z-tgfr1-2 1 5 unspecified role unspecified role neutral component neutral component region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig S5B. Immunofluorescence staining (IFS) also detected diffuse staining of both 14-3-3z and TGFBR1 proteins both in the cytosol and on the cell membrane. 1433z-tgfr1-3 1 5 unspecified role unspecified role neutral component neutral component region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig S9B. DCIS (Ductal Carcinoma In Situ) samples showed that co-overexpression of 14-3-3z and TGFBR1 significantly (p < 0.05) correlated with (at least two) EMT marker alterations. I assummed antibodies used were as stated in Supplementary Expt Proc TABLE for antibodies. This would imply the HA tag was used to detect 14-3-3-z, as no other ab is used for IFS to detect 14-3-3z. rac1-pak1-5 3 6 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait pbd Protein binding domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 physical association physical association Precipitation of activated (GTP bound) Rac1 from nutrient starved ErbB2-overexpressing MCF10A cells stimulated with MCF10A complete media. Fig S3A.