Figure 3
The mutation of K645, K646, R648 and R650 disrupts CIN85-Cbl interaction and inhibits EGFR degradation. (A) Interaction of CIN85 mutants with c-Cbl. HA-tagged c-Cbl and Flag-tagged CIN85 mutants were co-expressed in HEK293 cells and HA-Cbl was immunoprecipitated by anti-HA antibody (IP: α-HA-Cbl). CIN85 mutants were detected by immunoblot with anti-Flag antibody (IB). (B) Interaction of CIN85 mutants with endophilins. CIN85 mutants were expressed in COS7 cells and endogenous endophilins were immunoprecipitated by anti-endophilin antibody. (C) Interaction of EGFR and c-Cbl in the presence of CIN85 mutants. COS7 cells co-expressing CIN85 mutant and HA-Cbl were serum starved and stimulated with 25 ng/ml EGF for 30 minutes. HA-Cbl was immunoprecipitated by anti-HA antibody. CIN85 mutants and EGFR were detected by immunoblot with anti-Flag antibody and anti-EGFR antibody respectively. (D) Inhibition of EGFR downregulation by CIN85-ΔCC or CIN85-A4m. COS7 cells transfected with CIN85, CIN85-ΔCC, CIN85-A4m, CIN85-B4m or blank vector were serum starved and stimulated with 25 ng/ml EGF for indicated times (0, 15, 30, 60, 120, 180 min). Cells were harvested and EGFR was detected by Western blot. Tubulin was used as loading control. (E) Image gray statistical plot of EGFR downregulation in Figure 3D. Each gray level of EGFR electrophoretic band was normalized against relative EGFR level at 0 min. The final data were averaged from two independent western blot assays.