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/ Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Mol. Cell. Neurosci. (1044-7431) 2002 imex curation Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp A human brain cDNA library was used. orchard Only protein-protein interactions 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 Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp Accepted 2007-JAN-23 by ORCHARD. A human brain cDNA library was used. Mol. Cell. Neurosci. (1044-7431) 2002 orchard Only protein-protein interactions imex curation Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Mol. Cell. Neurosci. (1044-7431) 2002 imex curation Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp A human brain cDNA library was used. orchard Only protein-protein interactions in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting western blot western blot Immuno blot Immuno blot Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer Is there a more specific interaction detection method for ligand overlay? GST-PCD17 protein was blotted onto a PVDF membrane and probed with the FLAG-tagged purified C-terminal region of MRG X, followed by Western blotting with an anti-FLAG M2 antibody. The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp Accepted 2007-JAN-23 by ORCHARD. A human brain cDNA library was used. Mol. Cell. Neurosci. (1044-7431) 2002 orchard Only protein-protein interactions imex curation Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Mol. Cell. Neurosci. (1044-7431) 2002 imex curation Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp A human brain cDNA library was used. orchard Only protein-protein interactions human-t98g Human caucasian glioblastoma line T98G t98g Human Caucasian glioblastoma T98G anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer Cell lysates of cells transiently transfected with PDC17 and FLAG-tagged MRG X were prepared, followed by immunoprecipitation with anti-FLAG antibody affinity resin and immunoblotting with anti-Purkinje cell antibodies (anti-Yo). The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp Accepted 2007-JAN-23 by ORCHARD. A human brain cDNA library was used. Mol. Cell. Neurosci. (1044-7431) 2002 orchard Only protein-protein interactions imex curation Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Mol. Cell. Neurosci. (1044-7431) 2002 imex curation Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp A human brain cDNA library was used. orchard Only protein-protein interactions 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp Accepted 2007-JAN-23 by ORCHARD. A human brain cDNA library was used. Mol. Cell. Neurosci. (1044-7431) 2002 orchard Only protein-protein interactions imex curation Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Interaction of a paraneoplastic cerebellar degeneration-associated neuronal protein with the nuclear helix-loop-helix leucine zipper protein MRG X. Mol. Cell. Neurosci. (1044-7431) 2002 imex curation Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp A human brain cDNA library was used. orchard Only protein-protein interactions human-t98g Human caucasian glioblastoma line T98G t98g Human Caucasian glioblastoma T98G fluorescence imaging fluorescence microscopy fluorescence imaging predetermined predetermined participant predetermined Sakai K., Shirakawa T., Li Y., Kitagawa Y., Hirose G. Cancer - Interactions investigated in the context of cancer The sequences in Fig. 1 were compared to the exon sequence in the VEGA database in order to determine that the AS9 clone coded for amino acids 161-288 of full length MRG X. ksakai@kanazawa-med.ac.jp Accepted 2007-JAN-23 by ORCHARD. A human brain cDNA library was used. Mol. Cell. Neurosci. (1044-7431) 2002 orchard Only protein-protein interactions imex curation cdr2_human Cerebellar degeneration-related protein 2 Paraneoplastic cerebellar degeneration-associated antigen Major Yo paraneoplastic antigen CDR2 PCD17 protein protein human Homo sapiens Human MLAENLVEEFEMKEDEPWYDHQDLQQDLQLAAELGKTLLDRNTELEDSVQQMYTTNQEQLQEIEYLTKQVELLRQMNEQHAKVYEQLDVTARELEETNQKLVADSKASQQKILSLTETIECLQTNIDHLQSQVEELKSSGQGRRSPGKCDQEKPAPSFACLKELYDLRQHFVYDHVFAEKITSLQGQPSPDEEENEHLKKTVTMLQAQLSLERQKRVTMEEEYGLVLKENSELEQQLGATGAYRARALELEAEVAEMRQMLQSEHPFVNGVEKLVPDSLYVPFKEPSQSLLEEMFLTVPESHRKPLKRSSSETILSSLAGSDIVKGHEETCIRRAKAVKQRGISLLHEVDTQYSALKVKYEELLKKCQEEQDSLSHKAVQTSRAAAKDLTGVNAQSEPVASGWELASVNPEPVSSPTTPPEYKALFKEIFSCIKKTKQEIDEQRTKYRSLSSHS A7615F2C5AE08D2C o14596_human protein protein human Homo sapiens Human MLPAATEVQHRLQGQKDMVWKWMPLLLLLVCVATMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPACSATSSRTPVSMSAHQPGALDPAGESELAAKNASWMCPYAKSTVSAGGRIVTPPTRARATGTEDGTGPQELTSAQLGLSAAPLSPTSPLQLPFVKASGVTHTRSATTAEGAAAASRCGLLQPRATPTRKWRGSMLQPCM BA36B7DB95FC0E20 mo4l2_human Mortality factor 4-like protein 2 MORF4L2 KIAA0026 MRGX MORF-related gene X protein Transcription factor-like protein MRGX Protein MSL3-2 protein protein human Homo sapiens Human MSSRKQGSQPRGQQSAEEENFKKPTRSNMQRSKMRGASSGKKTAGPQQKNLEPALPGRWGGRSAENPPSGSVRKTRKNKQKTPGNGDGGSTSEAPQPPRKKRARADPTVESEEAFKNRMEVKVKIPEELKPWLVEDWDLVTRQKQLFQLPAKKNVDAILEEYANCKKSQGNVDNKEYAVNEVVAGIKEYFNVMLGTQLLYKFERPQYAEILLAHPDAPMSQVYGAPHLLRLFVRIGAMLAYTPLDEKSLALLLGYLHDFLKYLAKNSASLFTASDYKVASAEYHRKAL C9EFF517C76A565D assy_human Argininosuccinate synthase Citrulline--aspartate ligase ASS1 ASS protein protein human Homo sapiens Human MSSKGSVVLAYSGGLDTSCILVWLKEQGYDVIAYLANIGQKEDFEEARKKALKLGAKKVFIEDVSREFVEEFIWPAIQSSALYEDRYLLGTSLARPCIARKQVEIAQREGAKYVSHGATGKGNDQVRFELSCYSLAPQIKVIAPWRMPEFYNRFKGRNDLMEYAKQHGIPIPVTPKNPWSMDENLMHISYEAGILENPKNQAPPGLYTKTQDPAKAPNTPDILEIEFKKGVPVKVTNVKDGTTHQTSLELFMYLNEVAGKHGVGRIDIVENRFIGMKSRGIYETPAGTILYHAHLDIEAFTMDREVRKIKQGLGLKFAELVYTGFWHSPECEFVRHCIAKSQERVEGKVQVSVLKGQVYILGRESPLSLYNEELVSMNVQGDYEPTDATGFININSLRLKEYHRLQSKVTAK DIP protein P00966 original sequence version: 165 47CAD2373AE47E47 cdr2-morf4l2-2 4 8 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 1 The sequence given for MRGX in Fig. 1 shows the protein to be 288 aa (confirmed by comparison to the exon described in the VEGA database and the Uniprotkb entry)as the last codon ATG is the termination codon. However, in Fig. 2 the diagram shows MRG X as being 289 aa. This suggests that PCD17 physically interacts with MRG X in the two-hybrid system. The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. morf4l2-cdr2-1 1 6 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 1, 2 The sequence given for MRGX in Fig. 1 shows the protein to be 288 aa (confirmed by comparison to the exon described in the VEGA database and the Uniprotkb entry)as the last codon ATG is the termination codon. However, in Fig. 2 the diagram shows MRG X as being 289 aa. The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. As shown in Fig. 2, deletion of amino acids 120 to 141 of PCD17 corresponding to the leucine zipper domain resulted in loss of LacZ reporter activities, indicating that this region is required for binding to MRG X. morf4l2-cdr2-2 1 6 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 8 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 physical association physical association 1, 2 The sequence given for MRGX in Fig. 1 shows the protein to be 288 aa (confirmed by comparison to the exon described in the VEGA database and the Uniprotkb entry)as the last codon ATG is the termination codon. However, in Fig. 2 the diagram shows MRG X as being 289 aa. The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. deletion of amino acids 249 to 270 of MRG X containing the leucine zipper domain failed to induce LacZ reporter activities in interaction with PCD17. These results suggest that the leucine zipper domain is necessary for interaction with MRG X. cdr2-morf4l2 2 8 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal sufficient to bind sufficient binding region sufficient to bind range range certain certain sequence position certain 6 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction It is not clear what organism the MRG X was expressed in. 3 we then expressed the PCD17 peptides as a bacterial GST fusion protein separated by SDS-PAGE, and separated GST alone by SDS-PAGE as a control. These proteins were blotted onto a PVDF membrane and probed with the purified carboxyl terminal region of MRG X tagged with FLAG; Western blotting was then carried out using anti-FLAG M2 antibody. As shown in Fig. 3, a specific interaction was seen with GST-PCD17 The sequence given for MRGX in Fig. 1 shows the protein to be 288 aa (confirmed by comparison to the exon described in the VEGA database and the Uniprotkb entry)as the last codon ATG is the termination codon. However, in Fig. 2 the diagram shows MRG X as being 289 aa. The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. It is not specified which C-terminal construct of MRG X was used. cdr2-morf4l2-1 5 8 unspecified role unspecified role neutral component neutral component c-terminal BFP tag fluorescent prot tag fluorescent protein tag fluorescent prot tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 6 unspecified role unspecified role neutral component neutral component c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) colocalization colocalization 5 C and D The sequence given for MRGX in Fig. 1 shows the protein to be 288 aa (confirmed by comparison to the exon described in the VEGA database and the Uniprotkb entry)as the last codon ATG is the termination codon. However, in Fig. 2 the diagram shows MRG X as being 289 aa. The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. In some cotransfected cells, BFP-tagged MRG X was localized mainly in the nucleus and partially in the cytoplasm (A), and GFP-tagged PCD17 was expressed in the cytoplasm (B). In other cotransfected cells, BFP-tagged MRG X was expressed in the nucleus (C) , and GFP-tagged PCD17 was expressed mainly in the nucleus and partially in the cytoplasm (D). Q01850; Q15014 transfection Q01850; Q15014 overexpressed morf4l2-cdr2-3 3 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association 6 B The sequence given for MRGX in Fig. 1 shows the protein to be 288 aa (confirmed by comparison to the exon described in the VEGA database and the Uniprotkb entry)as the last codon ATG is the termination codon. However, in Fig. 2 the diagram shows MRG X as being 289 aa. The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. After immunoprecipitation using anti-FLAG antibody affinity resin, specific binding between PCD17 and MRG X in transfected cells was confirmed by im-munoblot with anti-Purkinje cell antibodies (anti-Yo) Q01850; Q15014 transfection cdr2- o14596 4 7 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. Seven of the other fifteen clones turned out to be ADH1, Eif-2, NADUF7, pI esterase, folate binding protein, arginosuccinate synthetase, and isocitrate dehydrogenase. cdr2- ass 4 6 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey physical association physical association The length of the CDR2 protein is given as 460 aa in Fig. 2, whereas the length of the Uniprotkb entry is 454 aa. Seven of the other fifteen clones turned out to be ADH1, Eif-2, NADUF7, pI esterase, folate binding protein, arginosuccinate synthetase, and isocitrate dehydrogenase.