FIGURE 4:

VCP controls levels of ubiquitination of Shoc2 and affects RAF-1/ERK1/2 activation. (A, B) Endogenous Shoc2 (A) or RAF-1 (B) was immunoprecipitated from denatured cell lysates of Cos1 cells treated with 5 µM of CB-5083 for 4 h. Shoc2 and RAF-1 ubiquitination was detected by immunoblotting using anti-ubiquitin (Ub) antibody. (C) Cos1 cells were serum-starved for 16 h, treated with 5 µM of CB-5083 for 4 h, and then stimulated with EGF (0.2 ng/ml) for 7, 15, and 30 min. Endogenous Shoc2 was precipitated from denatured cell lysates using anti-Shoc2 antibodies and its ubiquitination was detected with anti-ubiquitin (Ub) antibody. Immunoblots were analyzed with anti-Shoc2, -Ub, and -pERK1/2 antibodies. (D) Crude endosomal (CE) subcellular fractions were prepared from Cos1 cells treated with vehicle (DMSO) or 5 µM of CB-5083 for 4 h. Endosomal Shoc2 was immunoprecipitated and analyzed using the indicated antibodies. Rab5 was used as a loading control. (E) Shoc2 was precipitated from denatured endosomal fractions prepared in D using anti-Shoc2 antibodies. Shoc2 ubiquitination was detected with anti-Ub antibody. (F) Cos1 cells were transfected with GST-Shoc2 and YFP-RAF-1. At 48 h posttransfection, cells were serum-starved for 16 h and treated with vehicle (DMSO) or 5 µM of CB-5083 for 4 h and then stimulated with EGF (0.2 ng/ml) for 7 min. Shoc2 was immunoprecipitated using anti-GST antibody. Immunoblots were analyzed with anti-p-RAF-1 (S338), -RAF-1, -GST, and -pERK1/2 antibodies. Blots from the multiple experiments were analyzed. Bars represent the mean ± SE (n = 3) for pRAF-1 normalized to the value for GAPDH in arbitrary units (p < 0.01, by Student’s t test). The results in each panel are representative of those from three independent experiments.