Figure 2CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.A Histogram comparing the normalized CBL protein intensities resulting from AP-MS analysis of CD4+T cells from WT and CBLOSTmice prior to and after the specified stimulation times. For each time point, the distributions of the normalized intensities of the 9 data points (3 biological replicates x 3 technical replicates) corresponding to CD4+ T from wild-type (WT) and CBLOSTmice were compared using the adjusted p-value from a one-way ANOVA test (denoted as p(t)) and the value r(t) corresponding to the enrichment observed in the CBLOST samples as compared to WT samples. Different colors represent distinct biological replicates. CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.B Proteins are classified as CBL interactors according to their position in a volcano plot in which the mean p-valueis plotted against the corresponding mean enrichment for CBL(t)>(t)>OST versus WT samples. The time point shown corresponds to the unstimulated condition (t=0s). Mean was calculated using bootstrap resampling (see Results and Material and Methods). Proteins that displayed an enrichment r(t) greater than 2-fold and a corresponding p-value p(t) lower than a set threshold (see Material and Methods) in more than 90% of the bootstrap iterations were selected as specific partners (indicated in red). Names of four of the most significantly enriched proteins are indicated. Dashed lines represent thresholds on the p-value p*=10-3 and the enrichment r*=2 used to identify specific interactors of CBL. CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBLB (C) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow. CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBL (D) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow. CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.E Histograms showing the numbers of specific interactors binding to the CBL-OST or CBLB-OST proteins prior to and after TCR stimulation for different times and for which P(Detection) >0.9. CD4+T cells from wild-type (WT), CBLOST and CBLBOST mice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.F Pie charts showing the percentages of CBL and CBLB interactors detected in both the present study and the BioGRID public database. The size of the pie charts is proportional to the numbers of interactors per bait.