MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url&gt;http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. EMBO J. (0261-4189) 2006 imex curation Guthridge MA., Powell JA., Barry EF., Stomski FC., McClure BJ., Ramshaw H., Felquer FA., Dottore M., Thomas DT., To B., Begley CG., Lopez AF. Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Guthridge MA., Powell JA., Barry EF., Stomski FC., McClure BJ., Ramshaw H., Felquer FA., Dottore M., Thomas DT., To B., Begley CG., Lopez AF. EMBO J. (0261-4189) 2006 Only protein-protein interactions imex curation Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. EMBO J. (0261-4189) 2006 imex curation Guthridge MA., Powell JA., Barry EF., Stomski FC., McClure BJ., Ramshaw H., Felquer FA., Dottore M., Thomas DT., To B., Begley CG., Lopez AF. Only protein-protein interactions human-tf1 Human erythroleukaemia TF1 cells tf1 Human erythroleukaemia TF1 cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Guthridge MA., Powell JA., Barry EF., Stomski FC., McClure BJ., Ramshaw H., Felquer FA., Dottore M., Thomas DT., To B., Begley CG., Lopez AF. EMBO J. (0261-4189) 2006 Only protein-protein interactions imex curation Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. EMBO J. (0261-4189) 2006 imex curation Guthridge MA., Powell JA., Barry EF., Stomski FC., McClure BJ., Ramshaw H., Felquer FA., Dottore M., Thomas DT., To B., Begley CG., Lopez AF. Only protein-protein interactions human-monocyte Human mononuclear cells monocyte Monocyte, mononuclear cell anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Guthridge MA., Powell JA., Barry EF., Stomski FC., McClure BJ., Ramshaw H., Felquer FA., Dottore M., Thomas DT., To B., Begley CG., Lopez AF. EMBO J. (0261-4189) 2006 Only protein-protein interactions imex curation p85a_human Phosphatidylinositol 3-kinase regulatory subunit alpha PIK3R1 GRB1 Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha protein protein human Homo sapiens Human MSAEGYQYRALYDYKKEREEDIDLHLGDILTVNKGSLVALGFSDGQEARPEEIGWLNGYNETTGERGDFPGTYVEYIGRKKISPPTPKPRPPRPLPVAPGSSKTEADVEQQALTLPDLAEQFAPPDIAPPLLIKLVEAIEKKGLECSTLYRTQSSSNLAELRQLLDCDTPSVDLEMIDVHVLADAFKRYLLDLPNPVIPAAVYSEMISLAPEVQSSEEYIQLLKKLIRSPSIPHQYWLTLQYLLKHFFKLSQTSSKNLLNARVLSEIFSPMLFRFSAASSDNTENLIKVIEILISTEWNERQPAPALPPKPPKPTTVANNGMNNNMSLQDAEWYWGDISREEVNEKLRDTADGTFLVRDASTKMHGDYTLTLRKGGNNKLIKIFHRDGKYGFSDPLTFSSVVELINHYRNESLAQYNPKLDVKLLYPVSKYQQDQVVKEDNIEAVGKKLHEYNTQFQEKSREYDRLYEEYTRTSQEIQMKRTAIEAFNETIKIFEEQCQTQERYSKEYIEKFKREGNEKEIQRIMHNYDKLKSRISEIIDSRRRLEEDLKKQAAEYREIDKRMNSIKPDLIQLRKTRDQYLMWLTQKGVRQKKLNEWLGNENTEDQYSLVEDDEDLPHHDEKTWNVGSSNRNKAENLLRGKRDGTFLVRESSKQGCYACSVVVDGEVKHCVINKTATGYGFAEPYNLYSSLKELVLHYQHTSLVQHNDSLNVTLAYPVYAQQRR DIP protein P27986 original sequence version: 181 DIP protein P27986 original sequence version: 184 DIP protein P27986-1 original sequence version: 135 DIP protein P27986 original sequence version: 135 DIP protein P27986-1 original sequence version: 145 DIP protein P27986-1 original sequence version: 147 DIP protein P27986 original sequence version: 191 B9DAD8416C33140F il3rb_human Cytokine receptor common subunit beta CSF2RB IL3RB IL5RB GM-CSF/IL-3/IL-5 receptor common beta subunit CDw131 protein protein human Homo sapiens Human MVLAQGLLSMALLALCWERSLAGAEETIPLQTLRCYNDYTSHITCRWADTQDAQRLVNVTLIRRVNEDLLEPVSCDLSDDMPWSACPHPRCVPRRCVIPCQSFVVTDVDYFSFQPDRPLGTRLTVTLTQHVQPPEPRDLQISTDQDHFLLTWSVALGSPQSHWLSPGDLEFEVVYKRLQDSWEDAAILLSNTSQATLGPEHLMPSSTYVARVRTRLAPGSRLSGRPSKWSPEVCWDSQPGDEAQPQNLECFFDGAAVLSCSWEVRKEVASSVSFGLFYKPSPDAGEEECSPVLREGLGSLHTRHHCQIPVPDPATHGQYIVSVQPRRAEKHIKSSVNIQMAPPSLNVTKDGDSYSLRWETMKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHSMALPALEPSTRYWARVRVRTSRTGYNGIWSEWSEARSWDTESVLPMWVLALIVIFLTIAVLLALRFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC 3398E37FDB8F393A shc1_human SHC-transforming protein 1 SHC1 SHC SHCA Src homology 2 domain-containing-transforming protein C1 SHC-transforming protein A SHC-transforming protein 3 protein protein human Homo sapiens Human MDLLPPKPKYNPLRNESLSSLEEGASGSTPPEELPSPSASSLGPILPPLPGDDSPTTLCSFFPRMSNLRLANPAGGRPGSKGEPGRAADDGEGIVGAAMPDSGPLPLLQDMNKLSGGGGRRTRVEGGQLGGEEWTRHGSFVNKPTRGWLHPNDKVMGPGVSYLVRYMGCVEVLQSMRALDFNTRTQVTREAISLVCEAVPGAKGATRRRKPCSRPLSSILGRSNLKFAGMPITLTVSTSSLNLMAADCKQIIANHHMQSISFASGGDPDTAEYVAYVAKDPVNQRACHILECPEGLAQDVISTIGQAFELRFKQYLRNPPKLVTPHDRMAGFDGSAWDEEEEEPPDHQYYNDFPGKEPPLGGVVDMRLREGAAPGAARPTAPNAQTPSHLGATLPVGQPVGGDPEVRKQMPPPPPCPGRELFDDPSYVNVQNLDKARQAVGGAGPPNPAINGSAPRDLFDMKPFEDALRVPPPPQSVSMAEQLRGEPWFHGKLSRREAEALLQLNGDFLVRESTTTPGQYVLTGLQSGQPKHLLLVDPEGVVRTKDHRFESVSHLISYHMDNHLPIISAGSELCLQQPVERKL DIP protein P29353 original sequence version: 161 DIP protein P29353 original sequence version: 190 7EFA5CB185A548D1 sta5a_human Signal transducer and activator of transcription 5A STAT5A STAT5 protein protein human Homo sapiens Human MAGWIQAQQLQGDALRQMQVLYGQHFPIEVRHYLAQWIESQPWDAIDLDNPQDRAQATQLLEGLVQELQKKAEHQVGEDGFLLKIKLGHYATQLQKTYDRCPLELVRCIRHILYNEQRLVREANNCSSPAGILVDAMSQKHLQINQTFEELRLVTQDTENELKKLQQTQEYFIIQYQESLRIQAQFAQLAQLSPQERLSRETALQQKQVSLEAWLQREAQTLQQYRVELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLAGNGGPPEGSLDVLQSWCEKLAEIIWQNRQQIRRAEHLCQQLPIPGPVEEMLAEVNATITDIISALVTSTFIIEKQPPQVLKTQTKFAATVRLLVGGKLNVHMNPPQVKATIISEQQAKSLLKNENTRNECSGEILNNCCVMEYHQATGTLSAHFRNMSLKRIKRADRRGAESVTEEKFTVLFESQFSVGSNELVFQVKTLSLPVVVIVHGSQDHNATATVLWDNAFAEPGRVPFAVPDKVLWPQLCEALNMKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGLSVSWSQFNRENLPGWNYTFWQWFDGVMEVLKKHHKPHWNDGAILGFVNKQQAHDLLINKPDGTFLLRFSDSEIGGITIAWKFDSPERNLWNLKPFTTRDFSIRSLADRLGDLSYLIYVFPDRPKDEVFSKYYTPVLAKAVDGYVKPQIKQVVPEFVNASADAGGSSATYMDQAPSPAVCPQAPYNMYPQNPDHVLDQDGEFDLDETMDVARHVEELLRRPMDSLDSRLSPPAGLFTSARGSLS DIP protein P42229 original sequence version: 162 C64237295F88CFBE akt1_human RAC-alpha serine/threonine-protein kinase AKT1 PKB RAC RAC-PK-alpha Protein kinase B Proto-oncogene c-Akt Protein kinase B alpha protein protein human Homo sapiens Human MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKKQEEEEMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA DIP protein P31749 original sequence version: 184 DIP protein P31749 original sequence version: 168 DIP protein P31749 original sequence version: 102 DIP protein P31749 original sequence version: 110 DIP protein P31749 original sequence version: 160 DIP protein P31749 original sequence version: 136 DIP protein P31749 original sequence version: 175 DIP protein P31749 original sequence version: 201 DIP protein P31749 original sequence version: 166 DIP protein P31749 original sequence version: 178 DIP protein P31749-1 original sequence version: 194 DIP protein P31749 original sequence version: 106 DIP protein P31749 original sequence version: 141 6EAFF4F8AD436714 jak2_human Tyrosine-protein kinase JAK2 Janus kinase 2 JAK2 protein protein human Homo sapiens Human MGMACLTMTEMEGTSTSSIYQNGDISGNANSMKQIDPVLQVYLYHSLGKSEADYLTFPSGEYVAEEICIAASKACGITPVYHNMFALMSETERIWYPPNHVFHIDESTRHNVLYRIRFYFPRWYCSGSNRAYRHGISRGAEAPLLDDFVMSYLFAQWRHDFVHGWIKVPVTHETQEECLGMAVLDMMRIAKENDQTPLAIYNSISYKTFLPKCIRAKIQDYHILTRKRIRYRFRRFIQQFSQCKATARNLKLKYLINLETLQSAFYTEKFEVKEPGSGPSGEEIFATIIITGNGGIQWSRGKHKESETLTEQDLQLYCDFPNIIDVSIKQANQEGSNESRVVTIHKQDGKNLEIELSSLREALSFVSLIDGYYRLTADAHHYLCKEVAPPAVLENIQSNCHGPISMDFAISKLKKAGNQTGLYVLRCSPKDFNKYFLTFAVERENVIEYKHCLITKNENEEYNLSGTKKNFSSLKDLLNCYQMETVRSDNIIFQFTKCCPPKPKDKSNLLVFRTNGVSDVPTSPTLQRPTHMNQMVFHKIRNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETEVLLKVLDKAHRNYSESFFEAASMMSKLSHKHLVLNYGVCVCGDENILVQEFVKFGSLDTYLKKNKNCINILWKLEVAKQLAWAMHFLEENTLIHGNVCAKNILLIREEDRKTGNPPFIKLSDPGISITVLPKDILQERIPWVPPECIENPKNLNLATDKWSFGTTLWEICSGGDKPLSALDSQRKLQFYEDRHQLPAPKWAELANLINNCMDYEPDFRPSFRAIIRDLNSLFTPDYELLTENDMLPNMRIGALGFSGAFEDRDPTQFEERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEEHLRDFEREIEILKSLQHDNIVKYKGVCYSAGRRNLKLIMEYLPYGSLRDYLQKHKERIDHIKLLQYTSQICKGMEYLGTKRYIHRDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKVKEPGESPIFWYAPESLTESKFSVASDVWSFGVVLYELFTYIEKSKSPPAEFMRMIGNDKQGQMIVFHLIELLKNNGRLPRPDGCPDEIYMIMTECWNNNVNQRPSFRDLALRVDQIRDNMAG DIP protein O60674 original sequence version: 119 DIP protein O60674 original sequence version: 126 DIP protein O60674 original sequence version: 137 DIP protein O60674 original sequence version: 129 C30669EF1A7DA80C csf2rb-akt1 2 p85 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level GM-CSF bc 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Akt 8 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot certain certain sequence position certain certain certain sequence position certain resulting-ptm phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot certain certain sequence position certain certain certain sequence position certain resulting-ptm JAK2 9 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level STAT5 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association homomint mint A similar biphasic phosphorylation profile was observed in TF-1 cells, with Ser585 phosphorylation occurring at <10 pM and Tyr577 phosphorylation occurring at >10 pM (Figure 2D).In addition, the recruitment of 14-3-3 and p85 was also observed at GM-CSF concentrations below 10 pM (Figure 2D), while higher concentrations of GM-CSF (>10 pM) were necessary to promote betac tyrosine phosphorylation as well as the phosphorylation of JAK2, STAT5, ERK and Akt (Figure 2D). f2d csf2rb-akt1-1 3 Akt 8 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot certain certain sequence position certain certain certain sequence position certain resulting-ptm phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot certain certain sequence position certain certain certain sequence position certain resulting-ptm GM-CSF bc 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level STAT5 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f5a homomint mint we observed in cells derived from a patient with AML that Ser585 phosphorylation and recruitment of 14-3-3 were constitutive, while Tyr577 phosphorylation was normally regulated by cytokine (Figure 5A). csf2rb-pik3r1 1 betac peptide 5 peptide synthesis peptide synthesis predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined phosphorylated phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) certain certain sequence position certain certain certain sequence position certain prerequisite-ptm phosphorylated phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) certain certain sequence position certain certain certain sequence position certain prerequisite-ptm SHC 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level p85 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level association association f2a homomint mint we performed pulldown experiments to examine the ability of 14-3-3, Shc and p85 from cell lysates derived from HEK-293-T and CTL-EN cells to bind phospho-peptides encompassing Tyr577 and Ser585 of betac. The non-phospho-Tyr577/non-phospho-Ser585 control peptide did not precipitate p85, Shc or 14-3-3 from HEK-293T or CTL-EN lysates (Figure 2A, lane 1). The phospho-Tyr577/non-phospho-Ser585 peptide precipitated Shc and p85, but not 14-3-3 (Figure 2A, lane 2), while the non-phospho-Tyr577/phospho-Ser585 peptide precipitated 14-3-3 and p85 (Figure 2A, lane 3). Notably, while the phospho-Tyr577/phospho-Ser585 doubly phosphorylated peptide precipitated Shc and p85 (Figure 2A, lane 4), the binding of 14-3-3 was markedly reduced. This reduced binding was also observed when purified recombinant 14-3-3zeta was used in a peptide pulldown in a cell-free system (Figure 2A, recombinant 14-3-3). csf2rb-pik3r1-1 1 betac peptide 5 peptide synthesis peptide synthesis predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified phosphorylated phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) certain certain sequence position certain certain certain sequence position certain prerequisite-ptm tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined SHC 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level p85 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level association association f2a homomint mint we performed pulldown experiments to examine the ability of 14-3-3, Shc and p85 from cell lysates derived from HEK-293-T and CTL-EN cells to bind phospho-peptides encompassing Tyr577 and Ser585 of betac. The non-phospho-Tyr577/non-phospho-Ser585 control peptide did not precipitate p85, Shc or 14-3-3 from HEK-293T or CTL-EN lysates (Figure 2A, lane 1). The phospho-Tyr577/non-phospho-Ser585 peptide precipitated Shc and p85, but not 14-3-3 (Figure 2A, lane 2), while the non-phospho-Tyr577/phospho-Ser585 peptide precipitated 14-3-3 and p85 (Figure 2A, lane 3). Notably, while the phospho-Tyr577/phospho-Ser585 doubly phosphorylated peptide precipitated Shc and p85 (Figure 2A, lane 4), the binding of 14-3-3 was markedly reduced. This reduced binding was also observed when purified recombinant 14-3-3zeta was used in a peptide pulldown in a cell-free system (Figure 2A, recombinant 14-3-3). csf2rb-pik3r1-2 1 betac peptide 5 peptide synthesis peptide synthesis predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined phosphorylated phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) certain certain sequence position certain certain certain sequence position certain prerequisite-ptm p85 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level SHC 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level association association f2a homomint mint we performed pulldown experiments to examine the ability of 14-3-3, Shc and p85 from cell lysates derived from HEK-293-T and CTL-EN cells to bind phospho-peptides encompassing Tyr577 and Ser585 of betac. The non-phospho-Tyr577/non-phospho-Ser585 control peptide did not precipitate p85, Shc or 14-3-3 from HEK-293T or CTL-EN lysates (Figure 2A, lane 1). The phospho-Tyr577/non-phospho-Ser585 peptide precipitated Shc and p85, but not 14-3-3 (Figure 2A, lane 2), while the non-phospho-Tyr577/phospho-Ser585 peptide precipitated 14-3-3 and p85 (Figure 2A, lane 3). Notably, while the phospho-Tyr577/phospho-Ser585 doubly phosphorylated peptide precipitated Shc and p85 (Figure 2A, lane 4), the binding of 14-3-3 was markedly reduced. This reduced binding was also observed when purified recombinant 14-3-3zeta was used in a peptide pulldown in a cell-free system (Figure 2A, recombinant 14-3-3).