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 http://mint.bio.uniroma2.it/mint moharram-2016-2 in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Moharram SA., Chougule RA., Su X., Li T., Sun J., Zhao H., Rönnstrand L., Kazi JU. kazi.uddin@med.lu.se kazi.uddin@med.lu.se <kazi.uddin@med.lu.se>; Oncotarget 2016 Only protein-protein interactions imex curation imex curation moharram-2016-1 chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Moharram SA., Chougule RA., Su X., Li T., Sun J., Zhao H., Rönnstrand L., Kazi JU. kazi.uddin@med.lu.se kazi.uddin@med.lu.se <kazi.uddin@med.lu.se>; Oncotarget 2016 Only protein-protein interactions imex curation imex curation slap2_mouse Src-like-adapter 2 Sla2 Src-like adapter protein 2 protein protein mouse Mus musculus Mouse MGSLSSRGKTSSPSPSSSGPDQEPVSMQPERHKVTAVALGSFPAGEQARLSLRLGEPLTIISEDGDWWTVQSEVSGREYHMPSVYVAKVAHGWLYEGLSREKAEELLLLPGNPGGAFLIRESQTRRGCYSLSVRLSRPASWDRIRHYRIQRLDNGWLYISPRLTFPSLHALVEHYSELADGICCPLREPCVLQKLGPLPGKDTPPPVTVPTSSLNWKKLDRSLLFLEAPASGEASLLSEGLRESLSSYISLAEDPLDDA flt3_human Receptor-type tyrosine-protein kinase FLT3 FLK2 FL cytokine receptor Fetal liver kinase-2 FLT3 STK1 Fms-like tyrosine kinase 3 Stem cell tyrosine kinase 1 CD135 protein protein human Homo sapiens Human MPALARDGGQLPLLVVFSAMIFGTITNQDLPVIKCVLINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVEVDVSASITLQVLVDAPGNISCLWVFKHSSLNCQPHFDLQNRGVVSMVILKMTETQAGEYLLFIQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQGESCKEESPAVVKKEEKVLHELFGTDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNISFYATIGVCLLFIVVLTLLICHKYKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYDLKWEFPRENLEFGKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKEKADSSEREALMSELKMMTQLGSHENIVNLLGACTLSGPIYLIFEYCCYGDLLNYLRSKREKFHRTWTEIFKEHNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLHGNSFHSEDEIEYENQKRLEEEEDLNVLTFEDLLCFAYQVAKGMEFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDSNYVVRGNARLPVKWMAPESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYKLIQNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLADAEEAMYQNVDGRVSECPHTYQNRRPFSREMDLGLLSPQAQVEDS vav2_mouse Guanine nucleotide exchange factor VAV2 Vav2 protein protein mouse Mus musculus Mouse MEQWRQCGRWLIDCKVLPPNHRVVWPSAVVFDLAQALRDGVLLCQLLHNLSPGSIDLKDINFRPQMSQFLCLKNIRTFLKVCHDKFGLRNSELFDPFDLFDVRDFGKVISAVSRLSLHSIAQSKGIRPFPSEETAENDDDVYRSLEELADEHDLGEDIYDCVPCEDEGDDIYEDIIKVEVQQPMKMGMTEDDKRSCCLLEIQETEAKYYRTLEDIEKNYMGPLRLVLSPADMAAVFINLEDLIKVHHSFLRAIDVSMMAGGSTLAKVFLEFKERLLIYGEYCSHMEHAQSTLNQLLASREDFRQKVEECTLRVQDGKFKLQDLLVVPMQRVLKYHLLLKELLSHSADRPERQQLKEALEAMQDLAMYINEVKRDKETLKKISEFQCSIENLQVKLEEFGRPKIDGELKVRSIVNHTKQDRYLFLFDKVVIVCKRKGYSYELKEVIELLFHKMTDDPMHNKDIKKWSYGFYLIHLQGKQGFQFFCKTEDMKRKWMEQFEMAMSNIKPDKANANHHSFQMYTFDKTTNCKACKMFLRGTFYQGYLCTRCGVGAHKECLEVIPPCKMSSPADVDAPGAGPGPKMVAVQNYHGNPAPPGKPVLTFQTGDVIELLRGDPDSPWWEGRLVQTRKSGYFPSSSVKPCPVDGRPPTGRPPSREIDYTAYPWFAGNMERQQTDNLLKSHASGTYLIRERPAEAERFAISIKFNDEVKHIKVVEKDSWIHITEAKKFESLLELVEYYQCHSLKESFKQLDTTLKFPYKSRERTTSRASSRSPASCASYNFSFLSPQGLSFAPQAPSAPFWSVFTPRVIGTAVARYNFAARDMRELSLREGDVVKIYSRIGGDQGWWKGETNGRIGWFPSTYVEEEGVQ itk_mouse Tyrosine-protein kinase ITK/TSK T-cell-specific kinase IL-2-inducible T-cell kinase Kinase EMT Kinase TLK Itk Emt Tlk Tsk protein protein mouse Mus musculus Mouse MNNFILLEEQLIKKSQQKRRTSPSNFKVRFFVLTKASLAYFEDRHGKKRTLKGSIELSRIKCVEIVKSDISIPCHYKYPFQTLVYLQVVHDNYLLYVFAPDCESRQRWVLTLKEETRNNNSLVSKYHPNFWMDGRWRCCSQLEKPAVGCAPYDPSKNASKKPLPPTPEDNRRSFQEPEETLVIALYDYQTNDPQELALRCDEEYYLLDSSEIHWWRVQDKNGHEGYAPSSYLVEKSPNNLETYEWYNKSISRDKAEKLLLDTGKEGAFMVRDSRTPGTYTVSVFTKAIISENPCIKHYHIKETNDSPKRYYVAEKYVFDSIPLLIQYHQYNGGGLVTRLRYPVCSWRQKAPVTAGLRYGKWVIQPSELTFVQEIGSGQFGLVHLGYWLNKDKVAIKTIQEGAMSEEDFIEEAEVMMKLSHPKLVQLYGVCLEQAPICLVFEFMEHGCLSDYLRSQRGLFAAETLLGMCLDVCEGMAYLEKACVIHRDLAARNCLVGENQVIKVSDFGMTRFVLDDQYTSSTGTKFPVKWASPEVFSFSRYSSKSDVWSFGVLMWEVFSEGKIPYENRSNSEVVEDISTGFRLYKPRLASCHVYQIMNHCWKEKPEDRPPFSQLLSQLAEIAEAGL tec_mouse Tyrosine-protein kinase Tec Tec protein protein mouse Mus musculus Mouse MNFNTILEEILIKRSQQKKKTSLLNYKERLCVLPKSVLSYYEGRAEKKYRKGVIDISKIKCVEIVKNDDGVIPCQNKFPFQVVHDANTLYIFAPSPQSRDRWVKKLKEEIKNNNNIMIKYHPKFWADGSYQCCRQTEKLAPGCEKYNLFESSIRKTLPPAPEIKKRRPPPPIPPEEENTEEIVVAMYDFQATEAHDLRLERGQEYIILEKNDLHWWRARDKYGSEGYIPSNYVTGKKSNNLDQYEWYCRNTNRSKAEQLLRTEDKEGGFMVRDSSQPGLYTVSLYTKFGGEGSSGFRHYHIKETATSPKKYYLAEKHAFGSIPEIIEYHKHNAAGLVTRLRYPVSTKGKNAPTTAGFSYDKWEINPSELTFMRELGSGLFGVVRLGKWRAQYKVAIKAIREGAMCEEDFIEEAKVMMKLTHPKLVQLYGVCTQQKPIYIVTEFMERGCLLNFLRQRQGHFSRDMLLSMCQDVCEGMEYLERNSFIHRDLAARNCLVNEAGVVKVSDFGMARYVLDDQYTSSSGAKFPVKWCPPEVFNYSRFSSKSDVWSFGVLMWEIFTEGRMPFEKNTNYEVVTMVTRGHRLHRPKLATKYLYEVMLRCWQERPEGRPSLEDLLRTIDELVECEETFGR nck2_mouse Cytoplasmic protein NCK2 Nck2 Grb4 Growth factor receptor-bound protein 4 NCK adaptor protein 2 SH2/SH3 adaptor protein NCK-beta protein protein mouse Mus musculus Mouse MTEEVIVIAKWDYTAQQDQELDIRKNERLWLLDDSKTWWRVRNAANRTGYVPSNYVERKNSLKKGSLVKNLKDTLGLGKTRRKPSARDASPTPSTDAEYPANGSGADRIYDLNIPAFVKFAYVAEREDELSLVKGSRVTVMEKCSDGWWRGSFNGQIGWFPSNYVLEEADEAAAEAPSFLSLRRGTALSNGQGARVLHVVQTLYPFSSVTEEELSFEKGETMEVIEKPENDPEWWKCKNARGQVGLVPKNYVVVLSDGPALHPAHTPQISYTGPSASGRFAGREWYYGNVTRHQAECALNERGVEGDFLIRDSESSPSDFSVSLKASGRNKHFKVQLVDSVYCIGQRRFHSMDELVEHYKKAPIFTSEHGEKLYLVRALQ flt3-sla2-1 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr589 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 false phosphorylation increasing strength phostyr591 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-2 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr589;phostyr591 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 false certain certain sequence position certain certain certain sequence position certain false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2b Furthermore, a doubly phosphorylated peptide, pY589/pY591, showed dramatically increased affinity compared to either pY589 and pY591 alone (Figure 2B) flt3-sla2-4 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr566 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-5 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr572 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-6 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr599 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-7 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr726 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-8 3475726 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr793 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-9 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr919 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-10 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr955 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. flt3-sla2-11 3475726 N/A 3475728 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate flag tag 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 false chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 N/A 3475729 unspecified role unspecified role bait bait purified purified phostyr969 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 false phosphorylation increasing strength binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false f2a To identify the probable SLAP2 binding sites in FLT3, we used phosphopeptides corresponding to twelve FLT3 tyrosine phosphorylation sites. Multiple FLT3 phosphopeptides were able to pull-down SLAP2 from cell lysates (Figure 2A). Peptides corresponding to pY589 and pY591 displayed comparatively stronger association while other binding sites included pY599 and pY919. sla2-flt3-1 3475727 N/A 3475728 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag 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 false N/A 3475729 anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell physical association physical association false false false f1b COS-1 cells were transfected with plasmids for FLT3-WT or FLAG-tagged VAV2, SLAP2, CRK, ITK, TEC, NCK2, CRKL and empty vector. One day after transfection cells were stimulated with 100 ng/ml FL before lysis. Cell lysates were immunoprecipitated using 1 μg/ml anti-FLAG antibody and then analyzed by Western blotting. We observed a strong association of SLAP2 with ligand-stimulated FLT3 (Figure 1B). VAV2, ITK, TEC, and NCK2 displayed weaker interaction compared to that of SLAP2. vav2-flt3-1 3475727 N/A 3475730 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag 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 false N/A 3475729 anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell physical association physical association false false false f1b COS-1 cells were transfected with plasmids for FLT3-WT or FLAG-tagged VAV2, SLAP2, CRK, ITK, TEC, NCK2, CRKL and empty vector. One day after transfection cells were stimulated with 100 ng/ml FL before lysis. Cell lysates were immunoprecipitated using 1 μg/ml anti-FLAG antibody and then analyzed by Western blotting. We observed a strong association of SLAP2 with ligand-stimulated FLT3 (Figure 1B). VAV2, ITK, TEC, and NCK2 displayed weaker interaction compared to that of SLAP2. itk-flt3-1 3475727 N/A 3475731 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag 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 false N/A 3475729 anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell physical association physical association false false false f1b COS-1 cells were transfected with plasmids for FLT3-WT or FLAG-tagged VAV2, SLAP2, CRK, ITK, TEC, NCK2, CRKL and empty vector. One day after transfection cells were stimulated with 100 ng/ml FL before lysis. Cell lysates were immunoprecipitated using 1 μg/ml anti-FLAG antibody and then analyzed by Western blotting. We observed a strong association of SLAP2 with ligand-stimulated FLT3 (Figure 1B). VAV2, ITK, TEC, and NCK2 displayed weaker interaction compared to that of SLAP2. tec-flt3-1 3475727 N/A 3475729 anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell N/A 3475732 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag 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 false physical association physical association false false false f1b COS-1 cells were transfected with plasmids for FLT3-WT or FLAG-tagged VAV2, SLAP2, CRK, ITK, TEC, NCK2, CRKL and empty vector. One day after transfection cells were stimulated with 100 ng/ml FL before lysis. Cell lysates were immunoprecipitated using 1 μg/ml anti-FLAG antibody and then analyzed by Western blotting. We observed a strong association of SLAP2 with ligand-stimulated FLT3 (Figure 1B). VAV2, ITK, TEC, and NCK2 displayed weaker interaction compared to that of SLAP2. nck2-flt3-1 3475727 N/A 3475729 anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell N/A 3475733 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag 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 false physical association physical association false false false f1b COS-1 cells were transfected with plasmids for FLT3-WT or FLAG-tagged VAV2, SLAP2, CRK, ITK, TEC, NCK2, CRKL and empty vector. One day after transfection cells were stimulated with 100 ng/ml FL before lysis. Cell lysates were immunoprecipitated using 1 μg/ml anti-FLAG antibody and then analyzed by Western blotting. We observed a strong association of SLAP2 with ligand-stimulated FLT3 (Figure 1B). VAV2, ITK, TEC, and NCK2 displayed weaker interaction compared to that of SLAP2. sla2-flt3-2 3475727 N/A 3475728 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag 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 false N/A 3475729 anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell P36888:p.Lys644Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false f1c Association of SLAP2 with wild-type FLT3 was dependent on FL stimulation (Figure 1C). Oncogenic FLT3-ITD showed constitutive association with SLAP2 while a kinase-dead mutant of FLT3, FLT3-K644A, did not bind to SLAP2. FLt3 ligand sla2-flt3-4 3475727 N/A 3475728 anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell flag tag 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 false N/A 3475728 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false f1d Additionally, using HA- and FLAG-tagged SLAP2 plasmids we showed that SLAP2-HA and SLAP2-FLAG form a complex (Figure 1D). However, complex formation was not dependent on the expression of FLT3 or of ligand stimulation. FLT3 ligand flt3-sla2-3 3475727 N/A 3475728 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag 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 false N/A 3475729 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell P36888:p.Tyr591Phe mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P36888:p.[Tyr589Phe;Tyr591Phe] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P36888:p.Tyr589Phe mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false f2c a doubly phosphorylated peptide, pY589/pY591, showed dramatically increased affinity compared to either pY589 and pY591 alone (Figure 2B) and, similarly, mutations in these tyrosine residues significantly decreased association (Figure 2C). FLT3 ligand sla2-flt3-5 3475727 N/A 3475728 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell Q8R4L0:p.Arg120Glu mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false flag tag 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 false N/A 3475729 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell physical association physical association false false false f2d To explore whether the SLAP2 SH2 domain is involved in association with the phospho-tyrosine residues, we generated an SH2 domain mutant of SLAP2. The positively charged critical arginine residue (Arg 121) in the SH2 domain was replaced with a negatively charged glutamic acid residue. This mutation completely abolished FLT3 and SLAP2 interaction (Figure 2D) suggesting that a functional SLAP2 SH2 domain is required for the interaction with FLT3. FLT3 ligand