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/ Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Cancer cell 2015 imex curation Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer xdeng4@emory.edu 17-Sep-2015: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-h460 Homo sapiens non-small-cell lung cancer cell line H460 h460 human non-small-cell lung cancer cell line H460 fluorescence imaging fluorescence microscopy fluorescence imaging immunocytochemistry immunocytochemistry Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer Nu/Nu mice with NSCLC - H460 cell line xenografts. xdeng4@emory.edu Accepted 2015-JUL-21 by ORCHARD Cancer cell 2015 Only protein-protein interactions imex curation imex curation I have added ONE interaction for Supp fig 1B only + Data Proc comment - I have not (intentionally!) altered anything else. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Cancer cell 2015 imex curation Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer xdeng4@emory.edu 17-Sep-2015: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps tag fluorescence tag visualisation by fluorescence tag fluorescence Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer I have added TWO parameters to EBI-10896754, both on Bxl2 at Participant level- one on the "required to bind" Feature of Bcl2 (delta BH4 - Ki=655nM) and one for the WT - directly to the Bcl2 mol (Ki=3.3 nM. However I have also added an extra interaction (EBI-10896784) showing binding of Bcl2 directly to BDA-366 - the RHS side of the graph - which we would not normally annotate, where the tracer has been completely displaced - however here I can add the WT Ki=3.3 nM at interaction level. Not sure which is the best way to show this, and if this extra interaction is necessary -neither am I sure that the EBI-10896754 interaction is comprehensible as it stands! (A comment would be better-as I am not sure what adding a parameter to a Required to Bind feature is meant to show.) (alternatively I could add the 3.3Ki value to the main interaction for EBI-10896754, remove this value from WT - participant level, but leave the 655nM value on the Required to bind feature...and get rid of the extra interaction EBI-10896784. Competition Binding Assay:fluorescent peptide tracer and a nonfluorescent small-molecule inhibitor compete for binding to the target protein (Bcl-2). When the reaction mixture contains a small-molecule inhibitor, the tracer is displaced by the small-molecule inhibitor from the target protein and the emission signal becomes depolarized. The Bcl-XL interaction with the BAD peptide cannot be annotated as the Bcl-XL protein cannot be found on the stated suppliers web page (NeoBioSci). Similarly the Bfl-1/A1 interaction cannot be added as the origin of BFL-1 is ambiguous. xdeng4@emory.edu Accepted 2015-JUL-21 by ORCHARD Cancer cell 2015 Only protein-protein interactions imex curation imex curation I have added ONE interaction for Supp fig 1B only + Data Proc comment - I have not (intentionally!) altered anything else. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Cancer cell 2015 imex curation Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer xdeng4@emory.edu 17-Sep-2015: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa weight by bromide molecular weight estimation by bromide staining weight by bromide Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer have left Roles as "neutral" as there is no "probe". After incubation of reactants, 4 ul loading dye was added to a the sample, and electrophoresis was performed. Gels were stained with ethidium bromide solution, illuminated with UV light, and photographed. xdeng4@emory.edu Accepted 2015-JUL-21 by ORCHARD Cancer cell 2015 Only protein-protein interactions imex curation imex curation I have added ONE interaction for Supp fig 1B only + Data Proc comment - I have not (intentionally!) altered anything else. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. Cancer cell 2015 imex curation Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer xdeng4@emory.edu 17-Sep-2015: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-h460 Homo sapiens non-small-cell lung cancer cell line H460 h460 human non-small-cell lung cancer cell line H460 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Han B., Park D., Li R., Xie M., Owonikoko TK., Zhang G., Sica GL., Ding C., Zhou J., Magis AT., Chen ZG., Shin DM., Ramalingam SS., Khuri FR., Curran WJ., Deng X. Cancer - Interactions investigated in the context of cancer Results from Supplementarty figure 3D cannot be included as the antibody target "IP3R" could be one or more molecules. xdeng4@emory.edu Accepted 2015-JUL-21 by ORCHARD Cancer cell 2015 Only protein-protein interactions imex curation imex curation I have added ONE interaction for Supp fig 1B only + Data Proc comment - I have not (intentionally!) altered anything else. cisplatin cisplatin [PtCl2(NH3)2] Briplatin CDDP cis-[PtCl2(NH3)2] cis-DDP cis-diamminedichloroplatinum cis-Diamminedichloroplatinum(II) cis-diammineplatinum(II) dichloride cis-dichlorodiammineplatinum(II) cis-platin Cismaplat Cisplatin cisplatin cisplatine cisplatino cisplatinum Lederplatin Neoplatin Peyrone's chloride Peyrone's salt Platamine Platinex Platinol Randa (SP-4-2)-diamminedichloridoplatinum (SP-4-2)-diamminedichloroplatinum cis-diamminedichloridoplatinum(II) cis-diamminedichloroplatinum(II) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) b2l11_human Bcl-2-like protein 11 BCL2L11 BIM Bcl2-interacting mediator of cell death protein protein human Homo sapiens Human MAKQPSDVSSECDREGRQLQPAERPPQLRPGAPTSLQTEPQGNPEGNHGGEGDSCPHGSPQGPLAPPASPGPFATRSPLFIFMRRSSLLSRSSSGYFSFDTDRSPAPMSCDKSTQTPSPPCQAFNHYLSAMASMRQAEPADMRPEIWIAQELRRIGDEFNAYYARRVFLNNYQAAEDHPRMVILRLLRYIVRLVWRMH DIP protein O43521 original sequence version: 48 DIP protein O43521-1 original sequence version: 71 DIP protein O43521 original sequence version: 132 DIP protein O43521 original sequence version: 143 D75735E469CA6997 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 bak_human Bcl-2 homologous antagonist/killer Apoptosis regulator BAK Bcl-2-like protein 7 BAK1 BAK BCL2L7 CDN1 protein protein human Homo sapiens Human MASGQGPGPPRQECGEPALPSASEEQVAQDTEEVFRSYVFYRHQQEQEAEGVAAPADPEMVTLPLQPSSTMGQVGRQLAIIGDDINRRYDSEFQTMLQHLQPTAENAYEYFTKIATSLFESGINWGRVVALLGFGYRLALHVYQHGLTGFLGQVTRFVVDFMLHHCIARWIAQRGGWVAALNLGNGPILNVLVVLGVVLLGQFVVRRFFKS DIP protein Q16611 original sequence version: 74 DIP protein Q16611 original sequence version: 162 DIP protein Q16611 original sequence version: 97 DIP protein Q16611 original sequence version: 158 DIP protein Q16611 original sequence version: 151 DIP protein Q16611-1 original sequence version: 178 DIP protein Q16611-1 original sequence version: 180 DIP protein Q16611 original sequence version: 121 A2200FE72A46D04E puc19c pUC19c dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid synthetic construct synthetic construct 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 q07820-1 Induced myeloid leukemia cell differentiation protein Mcl-1 Bcl-2-like protein 3 Bcl-2-related protein EAT/mcl1 mcl1/EAT MCL1L MCL1 BCL2L3 MCL-1L protein protein human Homo sapiens Human MFGLKRNAVIGLNLYCGGAGLGAGSGGATRPGGRLLATEKEASARREIGGGEAGAVIGGSAGASPPSTLTPDSRRVARPPPIGAEVPDVTATPARLLFFAPTRRAAPLEEMEAPAADAIMSPEEELDGYEPEPLGKRPAVLPLLELVGESGNNTSTDGSLPSTPPPAEEEEDELYRQSLEIISRYLREQATGAKDTKPMGRSGATSRKALETLRRVGDGVQRNHETAFQGMLRKLDIKNEDDVKSLSRVMIHVFSDGVTNWGRIVTLISFGAFVAKHLKTINQESCIEPLAESITDVLVRTKRDWLVKQRGWDGFVEFFHVEDLEGGIRNVLLAFAGVAGVGAGLAYLIR mint D85821AC59275F1F bda-366 BDA-366 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) bak1-bcl2-2 2 10 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component N/A sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain N/A fluorescein fluorescein label fluorescein n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction Supp 1Ai Both the Wt and the BH4 deletion mutant appeared to bind to Bcl2 in the absence of BDA-366. bak1-bcl2-1 2 12 competitor competitor competitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role neutral component neutral component N/A required to bind necessary binding region required to bind range range range range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component N/A sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain N/A fluorescein fluorescein label fluorescein n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction Supp 1A/B The BH4 deletion mutant did not bind BDA-366 . BDA-366 directly binds to WT Bcl2 with high binding affinity (Ki = 3.3nM). Using a compound Bcl2 mutant D10A/N11A/R12A/E13A (AAAA) a Ki value of ~600nM was obtained, showing decreased BDA-366 binding. (results not shown). bcl2-bda_366-1 2 12 competitor competitor competitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role neutral component neutral component N/A required to bind necessary binding region required to bind range range range range ecoli Escherichia coli (strain K12) direct interaction direct interaction Supp 1Aiii 56yery-1 3 5 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 9 unspecified role unspecified role neutral component neutral component synthetic construct synthetic construct direct interaction direct interaction Supp 1K BDA-366 did not bind DNA 35a6twetr-1 4 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait association association 2G BDA-366 enhanced the Bcl2/Bax interaction in association with decreased Bcl2/Bim binding. 2435w4t-1 1 10 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component colocalization colocalization 6, Supp 5. BDA-366 bak1-bcl2-3 2 11 unspecified role unspecified role neutral component neutral component 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 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) 8 unspecified role unspecified role neutral component neutral component N/A sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain N/A fluorescein fluorescein label fluorescein n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 42 44 direct interaction direct interaction Supp 1B