Figure 3.
Figure 3. Cbl family E3 ubiquitin ligases are degraded in cells with reduced IFT20, leading to defects in termination of PDGFRα 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 PDGFRα (p-PDGFRα) 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).

Cbl family E3 ubiquitin ligases are degraded in cells with reduced IFT20, leading to defects in termination of PDGFRα 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 PDGFRα (p-PDGFRα) 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).

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