FIG 6.

HUWE1-mediated ubiquitination is required to modulate the ERK1/2 pathway. (A) HeLa cells were depleted of HUWE1, Shoc2, or both by siRNA. At 48 h posttransfection, the cells were serum starved for 16 h and stimulated with EGF (10 ng/ml) for 7 or 15 min. The expression of the indicated proteins was analyzed using specific antibodies. Multiple blots from the experiments whose results are exemplified in panel A were analyzed. Bars represent the mean values ± SDs (n = 3) for phosphorylated ERK1/2 (pERK1/2; 7 min of EGF treatment) normalized to the value for GAPDH in arbitrary units (phosphorylated ERK1/2/GAPDH). (B) Cos1 cells stably depleted of Shoc2 (Cos-LV1) were transiently transfected with HUWE1 siRNA. In Cos-SR cells, Shoc2 was rescued with Shoc2-tRFP. At 48 h posttransfection, the cells were serum starved for 16 h and stimulated with EGF (0.2 ng/ml) for 7 min. The expression of the indicated proteins was analyzed using specific antibodies. Multiple blots from the experiments for which the results are exemplified in panel B were analyzed. Bars represent the mean values ± SDs (n = 3) for phosphorylated ERK1/2 (7 min of EGF treatment) normalized to the value for GAPDH in arbitrary units (phosphorylated ERK1/2/GAPDH). The results in each panel are representative of those from three independent experiments. *, a proteolytic fragment of Shoc2-tRFP that is often detected by immunoblotting in cells expressing full-length Shoc2-tRFP. NT, nontargeting siRNA; tERK1/2, total ERK1/2; H, HUWE1 siRNA.