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/ Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors. Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors. Eur. J. Biochem. (0014-2956) 2000 imex curation Smit DJ., Smith AG., Parsons PG., Muscat GE., Sturm RA. Cancer - Interactions investigated in the context of cancer R.Sturm@mailbox.uq.edu.au 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Smit DJ., Smith AG., Parsons PG., Muscat GE., Sturm RA. Cancer - Interactions investigated in the context of cancer R.Sturm@mailbox.uq.edu.au Accepted 2007-DEC-20 by JYOTI. 22-Jan-2008: Contacted by JYOTI. Eur. J. Biochem. (0014-2956) 2000 Only protein-protein interactions imex curation Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors. Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors. Eur. J. Biochem. (0014-2956) 2000 imex curation Smit DJ., Smith AG., Parsons PG., Muscat GE., Sturm RA. Cancer - Interactions investigated in the context of cancer R.Sturm@mailbox.uq.edu.au 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Smit DJ., Smith AG., Parsons PG., Muscat GE., Sturm RA. Cancer - Interactions investigated in the context of cancer R.Sturm@mailbox.uq.edu.au Accepted 2006-NOV-20 by JYOTI. 22-Jan-2008: Contacted by JYOTI. Eur. J. Biochem. (0014-2956) 2000 Only protein-protein interactions imex curation Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors. Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors. Eur. J. Biochem. (0014-2956) 2000 imex curation Smit DJ., Smith AG., Parsons PG., Muscat GE., Sturm RA. Cancer - Interactions investigated in the context of cancer R.Sturm@mailbox.uq.edu.au 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-jeg3 Homo sapiens choriocarcinoma JEG3 jeg3 Human choriocarcinoma JEG3 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 Smit DJ., Smith AG., Parsons PG., Muscat GE., Sturm RA. Cancer - Interactions investigated in the context of cancer R.Sturm@mailbox.uq.edu.au Accepted 2006-NOV-20 by JYOTI. Eur. J. Biochem. (0014-2956) 2000 Only protein-protein interactions imex curation sox10_human Transcription factor SOX-10 SOX10 protein protein human Homo sapiens Human MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGTAAIQAHYKSAHLDHRHPGEGSPMSDGNPEHPSGQSHGPPTPPTTPKTELQSGKADPKRDGRSMGEGGKPHIDFGNVDIGEISHEVMSNMETFDVAELDQYLPPNGHPGHVSSYSAAGYGLGSALAVASGHSAWISKPPGVALPTVSPPGVDAKAQVKTETAGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHSGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPTHWEQPVYTTLSRP FAA1EC108D4DE6A1 po3f2_human POU domain, class 3, transcription factor 2 POU3F2 BRN2 OCT7 OTF7 Nervous system-specific octamer-binding transcription factor N-Oct-3 Brain-specific homeobox/POU domain protein 2 Octamer-binding transcription factor 7 Octamer-binding protein 7 protein protein human Homo sapiens Human MATAASNHYSLLTSSASIVHAEPPGGMQQGAGGYREAQSLVQGDYGALQSNGHPLSHAHQWITALSHGGGGGGGGGGGGGGGGGGGGGDGSPWSTSPLGQPDIKPSVVVQQGGRGDELHGPGALQQQHQQQQQQQQQQQQQQQQQQQQQRPPHLVHHAANHHPGPGAWRSAAAAAHLPPSMGASNGGLLYSQPSFTVNGMLGAGGQPAGLHHHGLRDAHDEPHHADHHPHPHSHPHQQPPPPPPPQGPPGHPGAHHDPHSDEDTPTSDDLEQFAKQFKQRRIKLGFTQADVGLALGTLYGNVFSQTTICRFEALQLSFKNMCKLKPLLNKWLEEADSSSGSPTSIDKIAAQGRKRKKRTSIEVSVKGALESHFLKCPKPSAQEITSLADSLQLEKEVVRVWFCNRRQKEKRMTPPGGTLPGAEDVYGGSRDTPPHHGVQTPVQ D470360894D788C6 pax3_human Paired box protein Pax-3 PAX3 HUP2 HuP2 protein protein human Homo sapiens Human MTTLAGAVPRMMRPGPGQNYPRSGFPLEVSTPLGQGRVNQLGGVFINGRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPKQVTTPDVEKKIEEYKRENPGMFSWEIRDKLLKDAVCDRNTVPSVSSISRILRSKFGKGEEEEADLERKEAEESEKKAKHSIDGILSERASAPQSDEGSDIDSEPDLPLKRKQRRSRTTFTAEQLEELERAFERTHYPDIYTREELAQRAKLTEARVQVWFSNRRARWRKQAGANQLMAFNHLIPGGFPPTAMPTLPTYQLSETSYQPTSIPQAVSDPSSTVHRPQPLPPSTVHQSTIPSNPDSSSAYCLPSTRHGFSSYTDSFVPPSGPSNPMNPTIGNGLSPQVMGLLTNHGGVPHQPQTDYALSPLTGGLEPTTTVSASCSQRLDHMKSLDSLPTSQSYCPPTYSTTGYSMDPVTGYQYGQYGQSKPWTF 8AFCA674E3ACB4FE tf2b_human Transcription initiation factor IIB GTF2B TF2B TFIIB General transcription factor TFIIB S300-II protein protein human Homo sapiens Human MASTSRLDALPRVTCPNHPDAILVEDYRAGDMICPECGLVVGDRVIDVGSEWRTFSNDKATKDPSRVGDSQNPLLSDGDLSTMIGKGTGAASFDEFGNSKYQNRRTMSSSDRAMMNAFKEITTMADRINLPRNIVDRTNNLFKQVYEQKSLKGRANDAIASACLYIACRQEGVPRTFKEICAVSRISKKEIGRCFKLILKALETSVDLITTGDFMSRFCSNLCLPKQVQMAATHIARKAVELDLVPGRSPISVAAAAIYMASQASAEKRTQKEIGDIAGVADVTIRQSYRLIYPRAPDLFPTDFKFDTPVDKLPQL DIP protein Q00403 original sequence version: 119 DIP protein Q00403 original sequence version: 181 9CC7E102526C2722 tbp_human TATA-box-binding protein TBP TF2D TFIID GTF2D1 TATA-box factor TATA-binding factor TATA sequence-binding protein Transcription initiation factor TFIID TBP subunit protein protein human Homo sapiens Human MDQNNSLPPYAQGLASPQGAMTPGIPIFSPMMPYGTGLTPQPIQNTNSLSILEEQQRQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQAVAAAAVQQSTSQQATQGTSGQAPQLFHSQTLTTAPLPGTTPLYPSPMTPMTPITPATPASESSGIVPQLQNIVSTVNLGCKLDLKTIALRARNAEYNPKRFAAVIMRIREPRTTALIFSSGKMVCTGAKSEEQSRLAARKYARVVQKLGFPAKFLDFKIQNMVGSCDVKFPIRLEGLVLTHQQFSSYEPELFPGLIYRMIKPRIVLLIFVSGKVVLTGAKVRAEIYEAFENIYPILKGFRKTT DIP protein P20226 original sequence version: 106 DIP protein P20226 original sequence version: 157 DIP protein P20226 original sequence version: 173 DIP protein P20226 original sequence version: 194 DIP protein P20226 original sequence version: 179 DIP protein P20226 original sequence version: 131 A61A578D972B970B ep300_human Histone acetyltransferase p300 Histone butyryltransferase p300 Histone crotonyltransferase p300 Protein propionyltransferase p300 EP300 P300 E1A-associated protein p300 Protein 2-hydroxyisobutyryltransferase p300 Protein lactyltransferas p300 protein protein human Homo sapiens Human MAENVVEPGPPSAKRPKLSSPALSASASDGTDFGSLFDLEHDLPDELINSTELGLTNGGDINQLQTSLGMVQDAASKHKQLSELLRSGSSPNLNMGVGGPGQVMASQAQQSSPGLGLINSMVKSPMTQAGLTSPNMGMGTSGPNQGPTQSTGMMNSPVNQPAMGMNTGMNAGMNPGMLAAGNGQGIMPNQVMNGSIGAGRGRQNMQYPNPGMGSAGNLLTEPLQQGSPQMGGQTGLRGPQPLKMGMMNNPNPYGSPYTQNPGQQIGASGLGLQIQTKTVLSNNLSPFAMDKKAVPGGGMPNMGQQPAPQVQQPGLVTPVAQGMGSGAHTADPEKRKLIQQQLVLLLHAHKCQRREQANGEVRQCNLPHCRTMKNVLNHMTHCQSGKSCQVAHCASSRQIISHWKNCTRHDCPVCLPLKNAGDKRNQQPILTGAPVGLGNPSSLGVGQQSAPNLSTVSQIDPSSIERAYAALGLPYQVNQMPTQPQVQAKNQQNQQPGQSPQGMRPMSNMSASPMGVNGGVGVQTPSLLSDSMLHSAINSQNPMMSENASVPSLGPMPTAAQPSTTGIRKQWHEDITQDLRNHLVHKLVQAIFPTPDPAALKDRRMENLVAYARKVEGDMYESANNRAEYYHLLAEKIYKIQKELEEKRRTRLQKQNMLPNAAGMVPVSMNPGPNMGQPQPGMTSNGPLPDPSMIRGSVPNQMMPRITPQSGLNQFGQMSMAQPPIVPRQTPPLQHHGQLAQPGALNPPMGYGPRMQQPSNQGQFLPQTQFPSQGMNVTNIPLAPSSGQAPVSQAQMSSSSCPVNSPIMPPGSQGSHIHCPQLPQPALHQNSPSPVPSRTPTPHHTPPSIGAQQPPATTIPAPVPTPPAMPPGPQSQALHPPPRQTPTPPTTQLPQQVQPSLPAAPSADQPQQQPRSQQSTAASVPTPTAPLLPPQPATPLSQPAVSIEGQVSNPPSTSSTEVNSQAIAEKQPSQEVKMEAKMEVDQPEPADTQPEDISESKVEDCKMESTETEERSTELKTEIKEEEDQPSTSATQSSPAPGQSKKKIFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDRFVYTCNECKHHVETRWHCTVCEDYDLCITCYNTKNHDHKMEKLGLGLDDESNNQQAAATQSPGDSRRLSIQRCIQSLVHACQCRNANCSLPSCQKMKRVVQHTKGCKRKTNGGCPICKQLIALCCYHAKHCQENKCPVPFCLNIKQKLRQQQLQHRLQQAQMLRRRMASMQRTGVVGQQQGLPSPTPATPTTPTGQQPTTPQTPQPTSQPQPTPPNSMPPYLPRTQAAGPVSQGKAAGQVTPPTPPQTAQPPLPGPPPAAVEMAMQIQRAAETQRQMAHVQIFQRPIQHQMPPMTPMAPMGMNPPPMTRGPSGHLEPGMGPTGMQQQPPWSQGGLPQPQQLQSGMPRPAMMSVAQHGQPLNMAPQPGLGQVGISPLKPGTVSQQALQNLLRTLRSPSSPLQQQQVLSILHANPQLLAAFIKQRAAKYANSNPQPIPGQPGMPQGQPGLQPPTMPGQQGVHSNPAMQNMNPMQAGVQRAGLPQQQPQQQLQPPMGGMSPQAQQMNMNHNTMPSQFRDILRRQQMMQQQQQQGAGPGIGPGMANHNQFQQPQGVGYPPQQQQRMQHHMQQMQQGNMGQIGQLPQALGAEAGASLQAYQQRLLQQQMGSPVQPNPMSPQQHMLPNQAQSPHLQGQQIPNSLSNQVRSPQPVPSPRPQSQPPHSSPSPRMQPQPSPHHVSPQTSSPHPGLVAAQANPMEQGHFASPDQNSMLSQLASNPGMANLHGASATDLGLSTDNSDLNSNLSQSTLDIH DIP protein Q09472 original sequence version: 193 DIP protein Q09472 original sequence version: 186 DIP protein Q09472 original sequence version: 107 DIP protein Q09472 original sequence version: 112 DIP protein Q09472 original sequence version: 181 DIP protein Q09472 original sequence version: 183 DIP protein Q09472 original sequence version: 136 DIP protein Q09472 original sequence version: 146 DIP protein Q09472 original sequence version: 165 DIP protein Q09472 original sequence version: 192 DIP protein Q09472 original sequence version: 200 DIP protein Q09472 original sequence version: 206 DIP protein Q09472 original sequence version: 194 DIP protein Q09472 original sequence version: 171 DIP protein Q09472 original sequence version: 142 DIP protein Q09472 original sequence version: 180 8E869E1F174A6FEB pou3f2-pou3f2 3 5 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role prey prey physical association physical association 2 deletion of the POU-homeodomain in GAL4 Brn-2 1�355 (POU-S/L) increased the interaction with the VP16 Brn-2 1�443 fulllength fusion protein relative to that initially found with GAL4 Brn-2 1�443 itself (Fig. 2B). This suggests that the POUhomeodomain may offer some steric hindrance to dimerization in this arrangement. Further C-terminal deletion of the POUhomeodomain and linker region in GAL4 Brn-2 1�336 (POU-S) resulted in loss of interaction with the VP16 Brn-2 1�443 full-length target. The POU-linker (336-353) region is required for homodimerization but not POU-homeodomain (353-411) when only one interactor is truncated. Deletions also suggest some requirement for the Brn-2 N-terminal region. Deletion of the POU-homeodomain in GAL4 Brn-2 1�355 increased the interaction with the VP16 Brn-2 1�443 full-length fusion protein relative to that found with GAL4 Brn-2 1�443 itself. ep300-pou3f2 3 9 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role prey prey physical association physical association 3 Only the results of interaction with GAL4 p300 1�149 are shown (Fig. 3) as this is the only portion of the p300 protein that demonstrated significant interaction with Brn-2. Neither the Brn-2 N-terminal region or the POU domain alone could reproduce the level of interaction shown by the full-length Brn-2. This suggested that intact Brn-2 was required for the interaction or that two domains of Brn-2 were involved in binding. tbp-pou3f2-1 3 5 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association 4 Full-length Brn-2 was able to interact with TBP in this assay system, however, in contrast to the Oct-1 and Oct-2 interaction it was not able to show that the Brn-2 187�443 POU domain alone was sufficient for this interaction. sox10-pou3f2 3 5 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait physical association physical association 5 A There was a sevenfold activation of reporter activity between full-length Sox10 and Brn-2 fusion proteins compared with VP16 alone and this level of interaction was lost with each of the C-terminal truncated Brn-2 proteins, indicating that an intact POU domain was required for interaction It appears in Fig. 5A that there was significant reporter activity with Brn-2 1-336 but this seems anomalous as there was not with Brn-2 1-355. The results indicate that the POU domain of Brn-2 is required for interaction with Sox10. sox10-sox10 3 4 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait physical association physical association 5 A It was notable that this level of interaction, although much less than the 70�200-fold seen between Brn-2 itself (Fig. 2), is similar to that seen between Sox10 itself (Gal4 Sox10: VP16 Sox10 pax3-sox10 3 6 unspecified role unspecified role bait bait 4 unspecified role unspecified role prey prey physical association physical association 5 B the interaction of Pax3 with Sox10 was also examined and shown to be of the same order of magnitude as Pax3 with Brn-2 pax3-pou3f2 3 5 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait physical association physical association 5 B there was a 10-fold activation of reporter activity between full-length Pax3 and the VP16 Brn-2 fusion proteins compared with VP16 alone, and again this level of interaction was lost in the each of the C-terminal truncated Brn-2 proteins indicating that an intact POU domain was required for interaction. The results indicate that the full POU domain of Brn-2 is required for interaction with Pax3. pou3f2-gtf2b 2 7 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 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 ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction 6 A Full-length GST-Brn-2 1�443 fusion protein was immobilized on glutathione agarose beads and incubated with 35S-radiolabelled full-length native, p300, TFIIB and TBP general transcription factors. Each of these proteins was found to be capable of binding to beads containing GST-Brn-2 but not with the GST protein alone pou3f2-tbp 2 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 ecobb Escherichia coli (strain B / BL21) 8 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 6 A Full-length GST-Brn-2 1�443 fusion protein was immobilized on glutathione agarose beads and incubated with 35S-radiolabelled full-length native, p300, TFIIB and TBP general transcription factors. Each of these proteins was found to be capable of binding to beads containing GST-Brn-2 but not with the GST protein alone pou3f2-ep300 2 9 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 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 ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction 6 A Full-length GST-Brn-2 1�443 fusion protein was immobilized on glutathione agarose beads and incubated with 35S-radiolabelled full-length native, p300, TFIIB and TBP general transcription factors. Each of these proteins was found to be capable of binding to beads containing GST-Brn-2 but not with the GST protein alone sox10-pax3 2 6 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 4 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 ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction 6 C when GST-Sox10 fusion protein was immobilized on glutathione agarose beads each of the 35S-radiolabelled Sox10, Pax3 and Brn-2 proteins bound selectively compared to immobolized GST protein alone confirming the direct interaction of each of these molecules. pou3f2-pax3 2 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 ecobb Escherichia coli (strain B / BL21) 6 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 6 C Testing of 35S-radiolabelled full-length native, Sox10, Pax3 and Brn-2 itself also showed each protein was capable of direct interaction with the immobilized GST-Brn-2 sox10-pou3f2-2 2 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 ecobb Escherichia coli (strain B / BL21) 4 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 6 C Testing of 35S-radiolabelled full-length native, Sox10, Pax3 and Brn-2 itself also showed each protein was capable of direct interaction with the immobilized GST-Brn-2 sox10-sox10-1 2 4 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 ecobb Escherichia coli (strain B / BL21) 4 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 6 C when GST-Sox10 fusion protein was immobilized on glutathione agarose beads each of the 35S-radiolabelled Sox10, Pax3 and Brn-2 proteins bound selectively compared to immobolized GST protein alone confirming the direct interaction of each of these molecules. sox10-pou3f2-1 2 4 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 ecobb Escherichia coli (strain B / BL21) 5 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 6 C when GST-Sox10 fusion protein was immobilized on glutathione agarose beads each of the 35S-radiolabelled Sox10, Pax3 and Brn-2 proteins bound selectively compared to immobolized GST protein alone confirming the direct interaction of each of these molecules. pou3f2-pou3f2-1 2 5 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 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 ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction 6 C Testing of 35S-radiolabelled full-length native, Sox10, Pax3 and Brn-2 itself also showed each protein was capable of direct interaction with the immobilized GST-Brn-2 1-443 fusion protein but not GST pou3f2-ep300-1 1 5 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 9 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 6 B In addition p300 and Brn-2 proteins were able to be coimmunoprecipitated when challenged with an anti-(Brn-2) Ig