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/ GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. J. Cell. Physiol. (0021-9541) 2007 imex curation Shu CW., Sun FC., Cho JH., Lin CC., Liu PF., Chen PY., Chang MD., Fu HW., Lai YK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer yklai@life.nthu.edu.tw 01-Feb-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Shu CW., Sun FC., Cho JH., Lin CC., Liu PF., Chen PY., Chang MD., Fu HW., Lai YK. Apoptosis - Interactions involving proteins with a function related to apoptosis yklai@life.nthu.edu.tw Accepted 2008-FEB-21 by JYOTI. 01-Feb-2008: Contacted by JYOTI. J. Cell. Physiol. (0021-9541) 2007 Only protein-protein interactions imex curation GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. J. Cell. Physiol. (0021-9541) 2007 imex curation Shu CW., Sun FC., Cho JH., Lin CC., Liu PF., Chen PY., Chang MD., Fu HW., Lai YK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer yklai@life.nthu.edu.tw 01-Feb-2008: Contacted by JYOTI. Only protein-protein interactions human-h460 Homo sapiens non-small-cell lung cancer cell line H460 h460 human non-small-cell lung cancer cell line H460 cosedimentation cosedimentation western blot western blot Immuno blot Immuno blot Shu CW., Sun FC., Cho JH., Lin CC., Liu PF., Chen PY., Chang MD., Fu HW., Lai YK. Apoptosis - Interactions involving proteins with a function related to apoptosis yklai@life.nthu.edu.tw Accepted 2008-FEB-21 by JYOTI. 01-Feb-2008: Contacted by JYOTI. J. Cell. Physiol. (0021-9541) 2007 Only protein-protein interactions imex curation GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. J. Cell. Physiol. (0021-9541) 2007 imex curation Shu CW., Sun FC., Cho JH., Lin CC., Liu PF., Chen PY., Chang MD., Fu HW., Lai YK. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer yklai@life.nthu.edu.tw 01-Feb-2008: Contacted by JYOTI. Only protein-protein interactions 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 Shu CW., Sun FC., Cho JH., Lin CC., Liu PF., Chen PY., Chang MD., Fu HW., Lai YK. Apoptosis - Interactions involving proteins with a function related to apoptosis yklai@life.nthu.edu.tw Accepted 2008-FEB-21 by JYOTI. 01-Feb-2008: Contacted by JYOTI. J. Cell. Physiol. (0021-9541) 2007 Only protein-protein interactions imex curation pten_human Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN Phosphatase and tensin homolog Mutated in multiple advanced cancers 1 PTEN MMAC1 TEP1 protein protein human Homo sapiens Human MTAIIKEIVSRNKRRYQEDGFDLDLTYIYPNIIAMGFPAERLEGVYRNNIDDVVRFLDSKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSYLLKNHLDYRPVALLFHKMMFETIPMFSGGTCNPQFVVCQLKVKIYSSNSGPTRREDKFMYFEFPQPLPVCGDIKVEFFHKQNKMLKKDKMFHFWVNTFFIPGPEETSEKVENGSLCDQEIDSICSIERADNDKEYLVLTLTKNDLDKANKDKANRYFSPNFKVKLYFTKTVEEPSNPEASSSTSVTPDVSDNEPDHYRYSDTTDSDPENEPFDEDQHTQITKV 75F97C3DD6802BA9 bip_human Endoplasmic reticulum chaperone BiP HSPA5 GRP78 Immunoglobulin heavy chain-binding protein 78 kDa glucose-regulated protein Binding-immunoglobulin protein Heat shock protein 70 family protein 5 Heat shock protein family A member 5 protein protein human Homo sapiens Human MKLSLVAAMLLLLSAARAEEEDKKEDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGRTWNDPSVQQDIKFLPFKVVEKKTKPYIQVDIGGGQTKTFAPEEISAMVLTKMKETAEAYLGKKVTHAVVTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNGVFEVVATNGDTHLGGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAVQKLRREVEKAKRALSSQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLDVCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYGSAGPPPTGEEDTAEKDEL DIP protein P11021 original sequence version: 127 DIP protein P11021 original sequence version: 137 DIP protein P11021 original sequence version: 185 59B7D8D85BC32A00 ch60_human 60 kDa heat shock protein, mitochondrial HSPD1 HSP60 Heat shock protein 60 60 kDa chaperonin Chaperonin 60 Mitochondrial matrix protein P1 P60 lymphocyte protein HuCHA60 protein protein human Homo sapiens Human MLRLPTVFRQMRPVSRVLAPHLTRAYAKDVKFGADARALMLQGVDLLADAVAVTMGPKGRTVIIEQSWGSPKVTKDGVTVAKSIDLKDKYKNIGAKLVQDVANNTNEEAGDGTTTATVLARSIAKEGFEKISKGANPVEIRRGVMLAVDAVIAELKKQSKPVTTPEEIAQVATISANGDKEIGNIISDAMKKVGRKGVITVKDGKTLNDELEIIEGMKFDRGYISPYFINTSKGQKCEFQDAYVLLSEKKISSIQSIVPALEIANAHRKPLVIIAEDVDGEALSTLVLNRLKVGLQVVAVKAPGFGDNRKNQLKDMAIATGGAVFGEEGLTLNLEDVQPHDLGKVGEVIVTKDDAMLLKGKGDKAQIEKRIQEIIEQLDVTTSEYEKEKLNERLAKLSDGVAVLKVGGTSDVEVNEKKDRVTDALNATRAAVEEGIVLGGGCALLRCIPALDSLTPANEDQKIGIEIIKRTLKIPAMTIAKNAGVEGSLIVEKIMQSSSEVGYDAMAGDFVNMVEKGIIDPTKVVRTALLDAAGVASLLTTAEVVVTEIPKEEKDPGMGAMGGMGGGMGGGMF DIP protein P10809 original sequence version: 142 E51E1BAD9615899C raf1_human RAF proto-oncogene serine/threonine-protein kinase RAF1 RAF Proto-oncogene c-RAF Raf-1 protein protein human Homo sapiens Human MEHIQGAWKTISNGFGFKDAVFDGSSCISPTIVQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTIGDSGVPALPSLTMRRMRESVSRMPVSSQHRYSTPHAFTFNTSSPSSEGSLSQRQRSTSTPNVHMVSTTLPVDSRMIEDAIRSHSESASPSALSSSPNNLSPTGWSQPKTPVPAQRERAPVSGTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVF DIP protein P04049 original sequence version: 117 DIP protein P04049 original sequence version: 106 DIP protein P04049 original sequence version: 114 DIP protein P04049 original sequence version: 141 DIP protein P04049 original sequence version: 142 DIP protein P04049 original sequence version: 147 DIP protein P04049-1 original sequence version: 136 DIP protein P04049 original sequence version: 136 DIP protein P04049 original sequence version: 153 DIP protein P04049 original sequence version: 150 DIP protein P04049 original sequence version: 164 DIP protein P04049 original sequence version: 172 DIP protein P04049-1 original sequence version: 172 DIP protein P04049 original sequence version: 184 DIP protein P04049 original sequence version: 188 DIP protein P04049 original sequence version: 195 EF821B5349711BC3 hspa5-raf1 1 7 unspecified role unspecified role prey prey human-h460 Homo sapiens non-small-cell lung cancer cell line H460 h460 human non-small-cell lung cancer cell line H460 5 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) physical association physical association Fig. 5 B His6-GRP78 was constructed and expressed in E. coli BL21 (DE3) pLys for a pull-down assay. The purity of His6-GRP78 was confirmed by silver staining and verified by Western blot using anti-His or anti-GRP78 antibodies (Fig. 5A). From the pull-down assay, we found that GRP78 interacted with Raf-1 in both untreated and TG-treated cells GRP78 interacted with Raf-1 in both untreated and TG-treated cells. In the presence of ATP, GRP78 bound to Raf-1 with a lower affinity, implying that ATP stimulated Raf-1 release from GRP78. hspa5-raf1-1 3 5 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey physical association physical association the interaction between GRP78 and Raf-1 in H460 cells was demonstrated by coimmunoprecipitation analysis Fig. 5 C raf1-hspa5 3 7 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association the interaction between GRP78 and Raf-1 in H460 cells was demonstrated by coimmunoprecipitation analysis Fig. 5 C hspa5-raf1-2 2 5 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig. 5 D GRP78 colocalized with Raf-1 mainly in the mitochondrial fractions Phosphorylation of Raf-1 at Ser338 and/or Tyr340 in mitochondria, reported to block apoptosis, was markedly diminished in AEBSF-pretreated cells during ER stress (caused by TG treatment). hspa5-pten 2 5 unspecified role unspecified role neutral component neutral component 4 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig. 5 D GRP78 colocalized with Raf-1 mainly in the mitochondrial fractions hspa5-raf1-3 2 7 unspecified role unspecified role neutral component neutral component 5 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig. 5 D GRP78 colocalized with Raf-1 mainly in the mitochondrial fractions The microsomal fraction contains the ER fraction.