Fig 2: 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).

Fig 3: Structural Requirements for Vav1-Cbl-b-ICN1 Complex Formation(A) CoIP of Vav1 proteins with Cbl-b in Jurkat cells ectopically expressing the indicated combinations of proteins (top). Amount of immunoprecipitated Cbl-b was assessed by reblotting the same filter with antibodies to Cbl-b (third panel from top). Expression of ectopic Vav1 proteins (fourth and fifth panels from top) and endogenous tubulin a (loading control, bottom panel) was determined by WB using aliquots of the TCLs used in the immunoprecipitation step.(B) Depiction of the Cbl-b mutants used in these experiments. Mutations are shown as open circles.(C) CoIP of Vav1 with indicated Cbl-b proteins (top) in Jurkat cells. Controls for the immunoprecipitation and expression of proteins were done as indicated in (A).(D) CoIP of Vav1 proteins with ICN1 in Jurkat cells ectopically expressing the indicated combinations of proteins (top). Amount of immunoprecipitated ICN1 was assessed by reblotting the same filter with antibodies to ICN1. Expression of ectopic Vav1 proteins and endogenous tubulin a (loading control) was determined as in (A). Asterisk marks the tubulin a band from the previous immunoblot of the same filter.(E) Depiction of ICN1 mutants used in these experiments. TM, transmembrane; NLS, nuclear localization signal; TAD, transactivation domain. The domain whose deletion leads to loss of Vav1 binding is shaded in light blue.(F) CoIP of Vav1 with ICN1 mutant proteins in Jurkat cells expressing the indicated combinations of proteins (top). Controls for the immunoprecipitation and expression of proteins were done as indicated in previous panels. Asterisks in the second and third panels from the top indicate the immunoglobulin G band of the antibody to ICN1 and the ICN1 band remaining from the previous immunoblot of the same filter, respectively.(G) CoIP of Cbl-b and indicated Vav1 mutant proteins with ICN1 in WT and VAV1-/- Jurkat cells. Controls for the immunoprecipitation and expression of proteins were done as indicated in previous panels. lo and hi refer to a low and a high exposure of the same film, respectively.(H) CoIP of Vav1 with ICN1 in WT and CBLB knockdown (clone #sh1) Jurkat cells. Controls for the immunoprecipitation and expression of proteins were done as in previous panels.(I) Summary of the interactions found in these experiments. Direct coIP and catalytic interactions are shown using black and gray arrows, respectively. Ub, ubiquitinylation.See also Figure S6.

Fig 4: IFT20 interacts with Cbl-b and c-Cbl, which is targeted to the cilium in PDGF-AA–stimulated cells. (a) Validation of the IFT20-c-Cbl interaction. HEK293T cells coexpressing GFP-IFT20 with either FLAG–tagged WT c-Cbl (FLAG–c-CblWT) or empty FLAG vector were subjected to FLAG IP, followed by WB analysis. (b and c) Reciprocal IPs of endogenous IFT20 or c-Cbl from HEK293T cells. (d) FLAG IP of HEK293T cell extracts expressing FLAG–c-CblWT or FLAG–tagged RING mutant (p.C381A) c-Cbl (FLAG–c-Cbl*RING). (e) IFT20 IP of HEK293T cell extracts expressing FLAG–c-CblWT or FLAG–c-Cbl*RING, followed by WB analysis. (f) IFM of WT NIH3T3 cells coexpressing GFP-tagged c-Cbl and FLAG–tagged IFT20. The dashed line indicates the Golgi complex, the arrow shows the primary cilium, and the asterisks identify the ciliary base. The nucleus was visualized with DAPI. (g) IFM of WT NIH3T3 cells expressing GFP-tagged c-Cbl. The primary cilium was marked with anti–ARL13B (arrow). The dashed line indicates the Golgi complex, and asterisk indicates the ciliary base. The nucleus was visualized with DAPI. The outline of the cells in panels f and g is highlighted with a dashed line. (h and i) IFM of WT NIH3T3 cells expressing either GFP-tagged c-Cbl (h) or stained with anti–c-Cbl (i). The Golgi complex (dashed line) was labeled with anti–GM130, and the nucleus was visualized with DAPI. (j) Localization of c-Cbl to the primary cilium in WT NIH3T3 cells, labeled with anti–Ac-tub (arrow), in WT NIH3T3 cells before and after stimulation with PDGF-AA. The asterisk marks the ciliary base. The nucleus was visualized with DAPI. (k) Localization of c-Cbl to the primary cilium in WT NIH3T3 cells and labeled with anti–ARL13B (arrow) in IMCD3 cells before and after stimulation with PDGF-AA. The asterisk marks the ciliary base. The nucleus was visualized with DAPI. (l) Quantification of relative levels of c-Cbl in the primary cilium shown in panel k. Fluorescence was normalized to background levels. For each of the three experiments, >15 cells were quantified. Error bars represent means ± SEM (n = 3).

Fig 5: Conditional gene silencing of IFT20 is associated with defects in termination of PDGFRα signaling. (a) Experimental setup to verify IFT20 silencing efficiency in NIH3T3shIFT20 cells upon treatment with Dox for given times. All cells were grown for 6 d. Asterisks indicate day of Dox addition. (b) WB analysis showing IFT20 expression in NIH3T3shIFT20 cells during 0–6 d of Dox treatment using the experimental setup outlined in panel a. (c–e) IFM images of growth-arrested NIH3T3shIFT20 cells treated without (− Dox) or with Dox (+ Dox) for 6 d, showing cellular localization of IFT20. Primary cilia (arrows) were labeled with anti–acetylated α-tubulin (Ac-tub) or anti-detyrosinated α-tubulin (Glu-tub), and the ciliary base/centrosome was labeled with anti–p150glued (asterisks). Nuclei (nu) were visualized with DIC microscopy or DAPI staining. Anti–Giantin was used to label the Golgi complex (dashed line). (f) Percentage of ciliated NIH3T3shIFT20 cells after Dox treatment, as indicated. Error bars represent means ± SEM (n = 3). (g) WB analysis of phosphorylation of PDGFRα (p-PDGFRα), AKT (p-AKT), and ERK1/2 (p-ERK1/2) upon stimulation with PDGF-AA for indicated times in growth-arrested NIH3T3shIFT20 cells treated with or without Dox treatment for 6 d. (h) Quantification of protein phosphorylations shown in panel g. Error bars represent means ± SEM (n = 3). (i) WB analysis of c-Cbl and Cbl-b levels in growth-arrested NIH3T3shIFT20 cells as well as in cells stably expressing siRNA-resistant, GFP-tagged IFT20 (NIH3T3shIFT20-Res) after Dox treatment for 6 d. (j) IFM analysis of NIH3T3shIFT20-Res cells with or without Dox treatment for 6 d. Cells were stained with antibodies against GFP and IFT20, and cell nuclei were visualized with DAPI staining. Primary cilia (arrows) were labeled with anti–Glu-Tub and anti–Ac-tub and are shown as shifted overlays. Quantification of ciliated NIH3T3shIFT20-Res cells; error bars represent means ± SEM (n = 3). (k) WB analysis of phosphorylation of PDGFRα (p-PDGFRα) and AKT (p-AKT) upon stimulation with 50 ng/ml PDGF-AA for indicated times in growth-arrested NIH3T3shIFT20 versus NIH3T3shIFT20-Res cells, both treated with Dox for 6 d. (l) Quantification of protein phosphorylations shown in panel k. Error bars represent means ± SEM (n = 3).