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/ Conserved signal peptide of Notch3 inhibits interaction with proteasome. Conserved signal peptide of Notch3 inhibits interaction with proteasome. Biochem. Biophys. Res. Commun. (0006-291X) 2007 Zhang Y., Jia L., Lee SJ., Wang MM. The SOS-rescue screen was performed to identify cytoplasmic binding partners that interact with cytoplasmic Notch3. This membrane-associated two-hybrid system makes use of the Ras pathway in yeast. When localized at the plasma membrane, the yeast Ras guanyl nucleotide exchange factor (RGEF) cdc25 stimulates GDP/GTP exchange on Ras and promotes downstream signalling events that ultimately lead to the cell growth. A mutant yeast strain harbouring the temperature sensitive cdc25-2 allele is still able to grow at 25�C but fails to grow at 36�C. However, the human RGEF (hSOS) when targeted to the plasma membrane efficiently complements the mutation, leading to cell growth at 36�C micwang@umich.edu 23-MAY-2007: Contacted by JYOTI. Mouse brain and mouse embryo cDNA. human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Zhang Y., Jia L., Lee SJ., Wang MM. The SOS-rescue screen was performed to identify cytoplasmic binding partners that interact with cytoplasmic Notch3. This membrane-associated two-hybrid system makes use of the Ras pathway in yeast. When localized at the plasma membrane, the yeast Ras guanyl nucleotide exchange factor (RGEF) cdc25 stimulates GDP/GTP exchange on Ras and promotes downstream signalling events that ultimately lead to the cell growth. A mutant yeast strain harbouring the temperature sensitive cdc25-2 allele is still able to grow at 25�C but fails to grow at 36�C. However, the human RGEF (hSOS) when targeted to the plasma membrane efficiently complements the mutation, leading to cell growth at 36�C micwang@umich.edu Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Mouse brain and mouse embryo cDNA. Biochem. Biophys. Res. Commun. (0006-291X) Biochem. Biophys. Res. Commun. (0006-291X) 2007 Conserved signal peptide of Notch3 inhibits interaction with proteasome. Conserved signal peptide of Notch3 inhibits interaction with proteasome. Biochem. Biophys. Res. Commun. (0006-291X) 2007 Zhang Y., Jia L., Lee SJ., Wang MM. The SOS-rescue screen was performed to identify cytoplasmic binding partners that interact with cytoplasmic Notch3. This membrane-associated two-hybrid system makes use of the Ras pathway in yeast. When localized at the plasma membrane, the yeast Ras guanyl nucleotide exchange factor (RGEF) cdc25 stimulates GDP/GTP exchange on Ras and promotes downstream signalling events that ultimately lead to the cell growth. A mutant yeast strain harbouring the temperature sensitive cdc25-2 allele is still able to grow at 25�C but fails to grow at 36�C. However, the human RGEF (hSOS) when targeted to the plasma membrane efficiently complements the mutation, leading to cell growth at 36�C micwang@umich.edu 23-MAY-2007: Contacted by JYOTI. Mouse brain and mouse embryo cDNA. 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 Zhang Y., Jia L., Lee SJ., Wang MM. The SOS-rescue screen was performed to identify cytoplasmic binding partners that interact with cytoplasmic Notch3. This membrane-associated two-hybrid system makes use of the Ras pathway in yeast. When localized at the plasma membrane, the yeast Ras guanyl nucleotide exchange factor (RGEF) cdc25 stimulates GDP/GTP exchange on Ras and promotes downstream signalling events that ultimately lead to the cell growth. A mutant yeast strain harbouring the temperature sensitive cdc25-2 allele is still able to grow at 25�C but fails to grow at 36�C. However, the human RGEF (hSOS) when targeted to the plasma membrane efficiently complements the mutation, leading to cell growth at 36�C micwang@umich.edu Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Mouse brain and mouse embryo cDNA. Biochem. Biophys. Res. Commun. (0006-291X) Biochem. Biophys. Res. Commun. (0006-291X) 2007 psa1_mouse Proteasome subunit alpha type-1 Proteasome component C2 Macropain subunit C2 Multicatalytic endopeptidase complex subunit C2 Proteasome nu chain Psma1 protein protein mouse Mus musculus Mouse MFRNQYDNDVTVWSPQGRIHQIEYAMEAVKQGSATVGLKSKTHAVLVALKRAQSELAAHQKKILHVDNHIGISIAGLTADARLLCNFMRQECLDSRFVFDRPLPVSRLVSLIGSKTQIPTQRYGRRPYGVGLLIAGYDDMGPHIFQTCPSANYFDCRAMSIGARSQSARTYLERHMSEFMECNLDELVKHGLRALRETLPAEQDLTTKNVSIGIVGKDLEFTIYDDDDVSPFLDGLEERPQRKAQPSQAAEEPAEKADEPMEH DIP protein Q9R1P4 original sequence version: 65 CC791C56AFBA8043 notc3_human Neurogenic locus notch homolog protein 3 NOTCH3 protein protein human Homo sapiens Human MGPGARGRRRRRRPMSPPPPPPPVRALPLLLLLAGPGAAAPPCLDGSPCANGGRCTQLPSREAACLCPPGWVGERCQLEDPCHSGPCAGRGVCQSSVVAGTARFSCRCPRGFRGPDCSLPDPCLSSPCAHGARCSVGPDGRFLCSCPPGYQGRSCRSDVDECRVGEPCRHGGTCLNTPGSFRCQCPAGYTGPLCENPAVPCAPSPCRNGGTCRQSGDLTYDCACLPGFEGQNCEVNVDDCPGHRCLNGGTCVDGVNTYNCQCPPEWTGQFCTEDVDECQLQPNACHNGGTCFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGATCHDRVASFYCACPMGKTGLLCHLDDACVSNPCHEDAICDTNPVNGRAICTCPPGFTGGACDQDVDECSIGANPCEHLGRCVNTQGSFLCQCGRGYTGPRCETDVNECLSGPCRNQATCLDRIGQFTCICMAGFTGTYCEVDIDECQSSPCVNGGVCKDRVNGFSCTCPSGFSGSTCQLDVDECASTPCRNGAKCVDQPDGYECRCAEGFEGTLCDRNVDDCSPDPCHHGRCVDGIASFSCACAPGYTGTRCESQVDECRSQPCRHGGKCLDLVDKYLCRCPSGTTGVNCEVNIDDCASNPCTFGVCRDGINRYDCVCQPGFTGPLCNVEINECASSPCGEGGSCVDGENGFRCLCPPGSLPPLCLPPSHPCAHEPCSHGICYDAPGGFRCVCEPGWSGPRCSQSLARDACESQPCRAGGTCSSDGMGFHCTCPPGVQGRQCELLSPCTPNPCEHGGRCESAPGQLPVCSCPQGWQGPRCQQDVDECAGPAPCGPHGICTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGSCQDGVGSFSCSCLPGFAGPRCARDVDECLSNPCGPGTCTDHVASFTCTCPPGYGGFHCEQDLPDCSPSSCFNGGTCVDGVNSFSCLCRPGYTGAHCQHEADPCLSRPCLHGGVCSAAHPGFRCTCLESFTGPQCQTLVDWCSRQPCQNGGRCVQTGAYCLCPPGWSGRLCDIRSLPCREAAAQIGVRLEQLCQAGGQCVDEDSSHYCVCPEGRTGSHCEQEVDPCLAQPCQHGGTCRGYMGGYMCECLPGYNGDNCEDDVDECASQPCQHGGSCIDLVARYLCSCPPGTLGVLCEINEDDCGPGPPLDSGPRCLHNGTCVDLVGGFRCTCPPGYTGLRCEADINECRSGACHAAHTRDCLQDPGGGFRCLCHAGFSGPRCQTVLSPCESQPCQHGGQCRPSPGPGGGLTFTCHCAQPFWGPRCERVARSCRELQCPVGVPCQQTPRGPRCACPPGLSGPSCRSFPGSPPGASNASCAAAPCLHGGSCRPAPLAPFFRCACAQGWTGPRCEAPAAAPEVSEEPRCPRAACQAKRGDQRCDRECNSPGCGWDGGDCSLSVGDPWRQCEALQCWRLFNNSRCDPACSSPACLYDNFDCHAGGRERTCNPVYEKYCADHFADGRCDQGCNTEECGWDGLDCASEVPALLARGVLVLTVLLPPEELLRSSADFLQRLSAILRTSLRFRLDAHGQAMVFPYHRPSPGSEPRARRELAPEVIGSVVMLEIDNRLCLQSPENDHCFPDAQSAADYLGALSAVERLDFPYPLRDVRGEPLEPPEPSVPLLPLLVAGAVLLLVILVLGVMVARRKREHSTLWFPEGFSLHKDVASGHKGRREPVGQDALGMKNMAKGESLMGEVATDWMDTECPEAKRLKVEEPGMGAEEAVDCRQWTQHHLVAADIRVAPAMALTPPQGDADADGMDVNVRGPDGFTPLMLASFCGGALEPMPTEEDEADDTSASIISDLICQGAQLGARTDRTGETALHLAARYARADAAKRLLDAGADTNAQDHSGRTPLHTAVTADAQGVFQILIRNRSTDLDARMADGSTALILAARLAVEGMVEELIASHADVNAVDELGKSALHWAAAVNNVEATLALLKNGANKDMQDSKEETPLFLAAREGSYEAAKLLLDHFANREITDHLDRLPRDVAQERLHQDIVRLLDQPSGPRSPPGPHGLGPLLCPPGAFLPGLKAAQSGSKKSRRPPGKAGLGPQGPRGRGKKLTLACPGPLADSSVTLSPVDSLDSPRPFGGPPASPGGFPLEGPYAAATATAVSLAQLGGPGRAGLGRQPPGGCVLSLGLLNPVAVPLDWARLPPPAPPGPSFLLPLAPGPQLLNPGTPVSPQERPPPYLAVPGHGEEYPAAGAHSSPPKARFLRVPSEHPYLTPSPESPEHWASPSPPSLSDWSESTPSPATATGAMATTTGALPAQPLPLSVPSSLAQAQTQLGPQPEVTPKRQVLA DIP protein Q9UM47 original sequence version: 162 3E70EC12A59CD638 notc3-psma1 2 3 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1236406]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 1342-1342, sequence length 263 [EBI-1236406]33-1342 4 unspecified role unspecified role bait bait physical association physical association We performed a genetic screen in yeast to identify proteins that interact with Notch3 in the cytoplasm, using the yeast SOS-rescue screen [11]. The most frequently identified gene encoded PSMA1, a subunit of the 20S proteasome complex. notc3-psma1-1 1 3 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 4 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 physical association physical association To determine whether PSMA1 and Notch3 could interact in mammalian cells, we performed immunoprecipitations in transfected 293 cells. Notch3 specifically immunoprecipitated with PSMA1, suggesting that the two proteins bind in mammalian cel 4A