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To exclude the non-specific binding in pull down and obtain true stable interactome, cells expressing FLAG-tagged GFP with three biological replicates were untreated as the control. Then 3 HeLa-FLAG-GFP samples and 18 HeLa-FLAG-CBL samples were lysed and subjected to AP-MS. Similarly, To identify EGF induced temporal interactome of CBLB, cells expressing FLAG-tagged CBLB were untreated (0 min) or stimulated with EGF for 0.5 min, 2 min, 5 min, 10 min and 30 min, with three biological replicates at every time point. To exclude the non-specific binding in pull down and obtain true stable interactome, cells expressing FLAG-tagged GFP with three biological replicates were untreated as the control. Then 3 HeLa-FLAG-GFP samples and 18 HeLa-FLAG-CBLB samples were lysed and subjected to AP-MS."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Raw MS data were searched in MaxQuant software (version 1.5.5.1) with label-free quantification function. Digestion enzyme was set as trypsine/p. Tolerated missed cleavage was set as 2. The human database from Uniprot (downloaded at 2018-03-27 with 71772 entries) was used. Carbamidomethyl (C, +57 Da) was set as fix modification and Oxidation (M, +16 Da) was included as variable modification. \"Match between runs\" was selected for label-free quantification. The false discovery rate (FDR) was evaluated by searching a reverse database and was set as 0.01 for proteins and peptides. To determine specific stable interacting proteins of CBL or CBLB, Log2 transformed LFQ intensities of identified proteins in CBL (0 min) or CBLB (0 min) group were compared with those in GFP control group to filter non-specific binding proteins through Perseus software. Fold change and adjusted p values were calculated and presented as a volcano plot. Significantly changed proteins (with the threshold of FDR = 0.05 and S0 = 0.5) were considered as the stable interacting proteins of CBL or CBLB.  To determine EGF stimulation-induced temporal interactome of CBL or CBLB, protein groups in every time point following EGF stimulation (including 0.5 min, 2 min, 5 min, 10 min and 30 min) were compared with the no stimulation group (0 min) through Perseus software. At a given stimulation time point, significantly changed proteins (with the threshold of FDR = 0.05 and S0 = 0.5) were considered as the temporal interacting proteins of CBL or CBLB."],"omics_type":["Proteomics"],"labhead":["Ruijun Tian"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Department of Chemistry, College of Science,Southern University of Science and Technology,Shenzhen518055, China"],"species":["Homo Sapiens (human)"],"publication":["34134489 Liu J, Yang L, He A, Ke M, Fu C, Gao W, Xu R, Tian R. Stable and EGF-Induced Temporal Interactome Profiling of CBL and CBLB Highlights Their Signaling Complex Diversity. J Proteome Res. 2021 20(7):3709-3719 10.1021/acs.jproteome.1c00284"],"submitter_mail":["liujiebnu2009@163.com"],"submitter_affiliation":["Shenzhen Blood Center"],"submitter_country":["China"],"pubmed_abstract":["The epidermal growth factor receptor (EGFR) signal modulates cell proliferation, migration, and survival. Aberrant activation of EGFR constitutes the major cause of various cancers. Receptor ubiquitination and degradation mediated by CBL proteins play negative regulatory roles and control the intensity and duration of the signaling. With the construction of stable cell lines inducibly expressing FLAG-tagged CBL or CBLB, we identified 102 and 82 stable interacting proteins of CBL and CBLB, respectively, through the affinity purification followed by mass spectrometry (AP-MS) approach. Time-resolved profiling at six different time points combined with functional annotations of the temporal interactomes provides insights into the dynamic assembly of signal proteins upon EGFR signaling activation. Comparison between the interactomes of CBL and CBLB indicates their redundant but also complementary functions. Importantly, we validated the stable association of EPS15L1 and ITSN2 and temporal association of TNK2 to both CBL and CBLB through biochemical assays. Collectively, these results offer a useful resource for CBL and CBLB interactomes and highlight their prominent and diverse roles in the EGFR signaling network."],"pubmed_title":["Stable and EGF-Induced Temporal Interactome Profiling of CBL and CBLB Highlights Their Signaling Complex Diversity."],"pubmed_authors":["Liu Jie J, Yang Lijun L, He An A, Ke Mi M, Fu Changying C, Gao Weina W, Xu Ruilian R, Tian Ruijun R"],"additional_accession":[]},"is_claimable":false,"name":"Stable and temporal interactomes of CBL and CBLB","description":"CBL family proteins have been widely reported as negative regulators of various signal pathways, which determines the amplitude and duration of signaling response through mediating the ubiquitination and degradation of activated receptors. CBL and CBLB share high degrees of structural similarities, and they are more than just ubiquitin-protein ligases and contain rich interactive domains and motifs that nucleate the assembly of protein complex taking various cellular functions. However, proteome-wide interaction network mediated by CBL proteins was rarely reported. This study is aimed to explore CBL and CBLB-mediated interactomes, hoping to uncover their regulatory roles in EGFR signaling and provide new insight into the molecular events driving tumorigenesis. With the construction of stable HeLa cell lines inducibly expressing FLAG-tagged CBL or CBLB, we detected both stable interactomes and EGF stimulation-induced temporal interactomes of CBL and CBLB through AP-MS approach. Time-resolved profiling and functional annotations of the temporal interactomes sheds light on the dynamic assemble of signal proteins over the signal cascade. Moreover, comparison between interactomes of CBL and CBLB indicates the redundant but also complementary functions of them. This work offer a useful resource for CBL and CBLB interactome, highlighting their prominent and diverse roles in the EGFR signalsome.","dates":{"publication":"2026-07-14","submission":"2021-04-08"},"accession":"PXD025250","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:108018","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:112509","NEWT:46429","NEWT:475174","NEWT:111205","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:2736291","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:294671","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:1907535","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:166340","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:111225","NEWT:7719","NEWT:29073","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:2767846","NEWT:108061","NEWT:60711","NEWT:2020713","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:468822","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:37919","NEWT:342808","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:709964","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:156974","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:1538","NEWT:2065263","NEWT:199225","NEWT:177437","NEWT:717966","NEWT:9014","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:29022","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:1883426","NEWT:8165","NEWT:96794","NEWT:10401","NEWT:10886","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:1955249","NEWT:215451","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:329032","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:55307","NEWT:1122981","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:31345","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:141469","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:1578","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:291146","NEWT:29466","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:235455","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:911022","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:717989","NEWT:33952","NEWT:1255232","NEWT:11723","NEWT:445","NEWT:153009","NEWT:1004879","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:1871049","NEWT:215402","NEWT:1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