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/ http://www.ebi.ac.uk/ izawa-2000-1 Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid full dna sequence full identification by DNA sequencing full dna sequence Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 Only protein-protein interactions imex curation izawa-2000-4 Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 Only protein-protein interactions imex curation izawa-2000-2 Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. in vitro In vitro cosedimentation cosedimentation weight by comassie molecular weight estimation by coomasie staining weight by comassie Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. As the separation of K18 and recombinant Mrj protein produced as a GST fusion protein (GST-Mrj) on Coomassie Brilliant Blue-stained gel after SDS-polyacrylamide gel electrophoresis was not clear the samples were also detected with anti-GST antibody by immunoblotting to more clearly show the proportions of supernatant and precipitated GST-Mrj. Incubation in filament assembly buffer induced a rapid polymerization of K8 and K18. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 Only protein-protein interactions imex curation izawa-2000-3 Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip monoclonal immunost monoclonal antibody immunostaining monoclonal immunost Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 Only protein-protein interactions imex curation izawa-2000-5 Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip immunostaining immunostaining Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 Only protein-protein interactions imex curation k1c18_human Keratin, type I cytoskeletal 18 KRT18 CYK18 Keratin-18 PIG46 Cytokeratin-18 Cell proliferation-inducing gene 46 protein protein protein human Homo sapiens Human MSFTTRSTFSTNYRSLGSVQAPSYGARPVSSAASVYAGAGGSGSRISVSRSTSFRGGMGSGGLATGIAGGLAGMGGIQNEKETMQSLNDRLASYLDRVRSLETENRRLESKIREHLEKKGPQVRDWSHYFKIIEDLRAQIFANTVDNARIVLQIDNARLAADDFRVKYETELAMRQSVENDIHGLRKVIDDTNITRLQLETEIEALKEELLFMKKNHEEEVKGLQAQIASSGLTVEVDAPKSQDLAKIMADIRAQYDELARKNREELDKYWSQQIEESTTVVTTQSAEVGAAETTLTELRRTVQSLEIDLDSMRNLKASLENSLREVEARYALQMEQLNGILLHLESELAQTRAEGQRQAQEYEALLNIKVKLEAEIATYRRLLEDGEDFNLGDALDSSNSMQTIQKTTTRRIVDGKVVSETNDTKVLRH dnjb6_human DnaJ homolog subfamily B member 6 Heat shock protein J2 MSJ-1 HHDJ1 DNAJB6 HSJ2 MSJ1 MRJ protein protein human Homo sapiens Human MVDYYEVLGVQRHASPEDIKKAYRKLALKWHPDKNPENKEEAERKFKQVAEAYEVLSDAKKRDIYDKYGKEGLNGGGGGGSHFDSPFEFGFTFRNPDDVFREFFGGRDPFSFDFFEDPFEDFFGNRRGPRGSRSRGTGSFFSAFSGFPSFGSGFSSFDTGFTSFGSLGHGGLTSFSSTSFGGSGMGNFKSISTSTKMVNGRKITTKRIVENGQERVEVEEDGQLKSLTINGVADDDALAEERMRRGQNALPAQPAGLRPPKPPRPASLLRHAPHCLSEEEGEQDRPRAPGPWDPLASAAGLKEGGKRKKQKQREESKKKKSTKGNH fiba_human Fibrinogen alpha chain FGA protein protein human Homo sapiens Human MFSMRIVCLVLSVVGTAWTADSGEGDFLAEGGGVRGPRVVERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQDFTNRINKLKNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDNTYNRVSEDLRSRIEVLKRKVIEKVQHIQLLQKNVRAQLVDMKRLEVDIDIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAKDLLPSRDRQHLPLIKMKPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTSYGTGSETESPRNPSSAGSWNSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGSTGSWNSGSSGTGSTGNQNPGSPRPGSTGTWNPGSSERGSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGTRREYHTEKLVTSKGDKELRTGKEKVTSGSTTTTRRSCSKTVTKTVIGPDGHKEVTKEVVTSEDGSDCPEAMDLGTLSGIGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRGKSSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPVRDCDDVLQTHPSGTQSGIFNIKLPGSSKIFSVYCDQETSLGGWLLIQQRMDGSLNFNRTWQDYKRGFGSLNDEGEGEFWLGNDYLHLLTQRGSVLRVELEDWAGNEAYAEYHFRVGSEAEGYALQVSSYEGTAGDALIEGSVEEGAEYTSHNNMQFSTFDRDADQWEENCAEVYGGGWWYNNCQAANLNGIYYPGGSYDPRNNSPYEIENGVVWVSFRGADYSLRAVRMKIRPLVTQ k2c8_human Keratin, type II cytoskeletal 8 Keratin-8 KRT8 CYK8 Cytokeratin-8 Type-II keratin Kb8 protein protein human Homo sapiens Human MSIRVTQKSYKVSTSGPRAFSSRSYTSGPGSRISSSSFSRVGSSNFRGGLGGGYGGASGMGGITAVTVNQSLLSPLVLEVDPNIQAVRTQEKEQIKTLNNKFASFIDKVRFLEQQNKMLETKWSLLQQQKTARSNMDNMFESYINNLRRQLETLGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTEMENEFVLIKKDVDEAYMNKVELESRLEGLTDEINFLRQLYEEEIRELQSQISDTSVVLSMDNSRSLDMDSIIAEVKAQYEDIANRSRAEAESMYQIKYEELQSLAGKHGDDLRRTKTEISEMNRNISRLQAEIEGLKGQRASLEAAIADAEQRGELAIKDANAKLSELEAALQRAKQDMARQLREYQELMNVKLALDIEIATYRKLLEGEESRLESGMQNMSIHTKTTSGYAGGLSSAYGGLTSPGLSYSLGSSFGSGAGSSSFSRTSSSRAVVVKKIETRDGKLVSESSDVLPK 1433t_human 14-3-3 protein theta YWHAQ 14-3-3 protein tau 14-3-3 protein T-cell Protein HS1 protein protein human Homo sapiens Human MEKTELIQKAKLAEQAERYDDMATCMKAVTEQGAELSNEERNLLSVAYKNVVGGRRSAWRVISSIEQKTDTSDKKLQLIKDYREKVESELRSICTTVLELLDKYLIANATNPESKVFYLKMKGDYFRYLAEVACGDDRKQTIDNSQGAYQEAFDISKKEMQPTHPIRLGLALNFSVFYYEILNNPELACTLAKTAFDEAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDSAGEECDAAEGAEN hsp7c_human Heat shock cognate 71 kDa protein HSPA8 HSC70 HSP73 HSPA10 Heat shock 70 kDa protein 8 Lipopolysaccharide-associated protein 1 protein protein human Homo sapiens Human MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDAVVQSDMKHWPFMVVNDAGRPKVQVEYKGETKSFYPEEVSSMVLTKMKEIAEAYLGKTVTNAVVTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPTAAAIAYGLDKKVGAERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKHKKDISENKRAVRRLRTACERAKRTLSSSTQASIEIDSLYEGIDFYTSITRARFEELNADLFRGTLDPVEKALRDAKLDKSQIHDIVLVGGSTRIPKIQKLLQDFFNGKELNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERAMTKDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGILNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDEKQRDKVSSKNSLESYAFNMKATVEDEKLQGKINDEDKQKILDKCNEIINWLDKNQTAEKEEFEHQQKELEKVCNPIITKLYQSAGGMPGGMPGGFPGGGAPPSGGASSGPTIEEVD dnajb6-krt18 Y2H interaction between human dnajb6 and krt18 608941 N/A 608946 unspecified role unspecified role bait bait N/A 608947 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind full dna sequence full identification by DNA sequencing full dna sequence certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false fga-krt18-1 Y2H interaction between human FGA and krt18 608941 N/A 608948 unspecified role unspecified role prey prey N/A 608946 unspecified role unspecified role bait bait physical association physical association false false false krt8-krt18-1 Y2H interaction between human KRT8 and krt18 608941 N/A 608946 unspecified role unspecified role bait bait N/A 608949 unspecified role unspecified role prey prey physical association physical association false false false ywhaq-krt18-1 Y2H interaction between human YWHAQ and krt18 608941 N/A 608950 unspecified role unspecified role prey prey N/A 608946 unspecified role unspecified role bait bait physical association physical association false false false dnajb6-krt18-3 Y2H to determine which region of dnajb6 interacts with krt18 608942 N/A 608947 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false N/A 608946 unspecified role unspecified role prey prey physical association physical association false false false The interaction between dnajb6 and krt18 required the c-terminal of dnajb6. Figure 1B, 5B dnajb6-hspc70-1 Y2H to determine which region of dnajb6 interacts with hsp/c70 608942 N/A 608951 unspecified role unspecified role prey prey N/A 608947 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false The interaction between dnajb6 and hsp/c70 required the n-terminal of dnajb6. Figure 5B dnajb6-krt18-4 Y2H to determine which region of dnajb6 interacts with krt18 608942 N/A 608947 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false N/A 608946 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false 608977 608975 physical association physical association false false false The interaction between dnajb6 and krt18 required the c-terminal of dnajb6. Figure 1D dnajb6-krt18-1 Cosedimentation assay showing interaction between human dnajb6 and krt18/krt8 filaments 608943 N/A 608949 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) N/A 608947 unspecified role unspecified role neutral component neutral component region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag western blot western blot Immuno blot Immuno blot undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) N/A 608946 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association false false false Because Mrj interacted with K18, but not with K8, in the two-hybrid analysis, the Mrj sedimenting with K8/18 was thought to be associated with the K8/18 polymer via direct binding to K18. In addition, Mrj did not sediment with vimentin. Figure 2A, 2B krt8-krt18-2 Cosedimentation assay showing interaction between human krt18 and krt8 608943 N/A 608946 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) N/A 608949 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association false false false Krt18 interacted with K8 independantly of dnajb6. Figure 2A, 2B dnajb6-hspc70 Co-IP showing in-vivo interaction between human dnajb6, KRT18 and Hsp/c70 608944 N/A 608946 unspecified role unspecified role prey prey N/A 608947 unspecified role unspecified role bait bait human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 N/A 608951 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 association association false false false Mrj is associated with Hsp/c70 in-vivo. Figure 3B dnajb6-krt18-5 Colocalisation showing interaction between human dnajb6 and krt18/krt8 filaments 608945 N/A 608946 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 N/A 608947 unspecified role unspecified role bait bait human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 physical association physical association false false false Using anti-Mrj polyclonal and anti-krt18 monoclonal antibodies, it was discovered that dnajb6 (Mrj) colocalized with Krt18 around the perinuclear region. Figure 4.