Fig 2: Activity-Dependent Profiling c-Cbl E3 Ligase Activity and Activity-Based Proteomic Analyses of EGF-Stimulated Versus Unstimulated HEK293T Cells(A) Only c-Cbl (3 µM) preincubated with c-Src (1.5 µM) undergoes photoABP-Bpa31 (5 µM) crosslinking. Crosslinking is not observed when Cbl Y371 (3 µM), which cannot be phosphorylated at the activation site, is incubated with Src. Phosphorylation of Cbl results in reduced electrophoretic mobility.(B) Transient overexpression of GFP-Cbl and c-Src in mammalian HEK293 cells. Extracts were treated with photoABP-Bpa31 or the F62A control probe (5 µM). IB denotes immunoblot and the primary antibody used for detection is adjacent (i.e., anti-Cbl).(C) Immunoblot analysis of HEK293T extracts with either photoABP-Bpa31 or the photoABP-Bpa31 F62A control probe (10 µM). Blotting was carried out against the hexahistidine reporter tag present within the ABPs. Samples were irradiated for 60 min or irradiation was withheld.(D) Schematic depicting activity-based proteomic workflow with biotinylated photoABP-Bpa31.(E) Spectral counts obtained from ABP-profiled HEK293T cells. Search results were filtered against the PFAM domain term “RING” and only RING E3s with >2 spectral counts in any replicate experiment were plotted. Cells were serum-starved and either treated with or without EGF and with or without UV irradiation. Errors bar correspond to the standard error from two technical replicate LC-MS/MS analyses.

Fig 3: Cbl family E3 ubiquitin ligases are degraded in cells with reduced IFT20, leading to defects in termination of PDGFRa signaling. (a) WB analysis showing expression levels of c-Cbl and Cbl-b proteins in cycling (+ serum) and growth-arrested (- serum) NIH3T3 and RPE-1 cells. CDK1 marks cycling cells. (b) WB analysis of c-Cbl and Cbl-b in growth-arrested NIH3T3shIFT20 cells upon addition of Dox for 1–6 d. All cells were grown for 6 d, including 24 h of serum depletion before analysis. An unspecific band stained by the IFT20 antibody is labeled with an asterisk. (c) WB analysis of IFT20 in growth-arrested NIH3T3 and RPE-1 cells subjected to mock or siRNA(siR)-mediated silencing of c-Cbl. (d) Quantitative, real-time PCR analysis of relative Ift20, c-Cbl, and Cbl-b mRNA transcript levels in NIH3T3shIFT20 upon 6 d of Dox treatment, including 24 h of serum depletion. Error bars represent means ± SEM (n = 3). (e) WB analysis of c-Cbl and Cbl-b in cycling NIH3T3shIFT20 cells upon Dox treatment for 3 d in combination with lysosomal (NH4Cl) or proteasomal (MG-132) inhibitors for 24 h or 10 h, respectively. (f) IP of endogenous c-Cbl in NIH3T3shIFT20 cells with or without Dox for 3 d, followed by WB analysis with antibodies against mono- and polyubiquitin. (g) WB analysis of growth-arrested NIH3T3shIFT20 cells stably expressing FLAG–tagged WT or RING-mutant c-Cbl (NIH3T3shIFT20,FLAG–c-Cbl,WT and NIH3T3shIFT20,FLAG–c-Cbl*RING, respectively) upon 6 d of Dox treatment. (h) FLAG IP of protein lysates from NIH3T3shIFT20,FLAG–c-Cbl*RING cells transiently expressing GFP-tagged c-Cbl RING mutant (GFP-c-Cbl*RING) after treatment with Dox for 4 d. Experiments presented in panels a–c and e–h were repeated at least three times, and results from representative experiments are shown. (i) WB analysis of phosphorylation of PDGFRa (p-PDGFRa) and AKT (p-AKT) upon stimulation with 50 ng/ml PDGF-AA for indicated times in NIH3T3 cells subjected to siRNA (siR)-mediated silencing of c-Cbl, Cbl-b, or both for 72 h, including serum depletion for the last 24 h before stimulation to induce growth arrest. (j) Quantification of protein phosphorylations shown in panel i. Error bars represent means ± SEM (n = 3).

Fig 4: Activation of SFKs is not involved in the process by which the depletion of Csk increases the protein level of c-Cbl. Western blot analysis with antibodies against the indicated proteins. (a) Samples were prepared as described in Fig. 2(b). Samples were prepared as described in Fig. 3. Full-length blots are presented in Fig. S11.

Fig 5: No relationships between the protein level of LynA and that of E-cadherin or vimentin. HCT116 (a), HeLa S3 (b), DLD1 (c), RKO (d), MDA-MB231 (e) and MCF-7 (f) cells were transfected with siRNA for Csk (lane 2), a combination of siRNAs for Csk, c-Cbl and Cbl-b (lane 3) or the control siRNA (lane 1) and then cultured for 48 h and analysed by Western blotting with antibodies against the indicated proteins. E-cadherin was not detected (N.D.) in HeLa S3, RKO and MDA-MB231 cells. Vimentin was N.D. in HCT116, DLD1, RKO and MCF-7 cells. Full-length blots are presented in Fig. S12.