Figure 6.

Different signaling downstream of the EGFR in cells depleted of Tsg101 or hVps24. (A) HeLa cells treated with scrambled RNA, siRNA against Tsg101, or siRNA against hVps24 were plated out in one six-well dish each and starved for 3 h in the presence of cycloheximide. All wells except one in each dish were given a 5-min pulse of EGF and then chased in normal medium supplied with cycloheximide for the indicated times. The cells were then lysed and analyzed by SDS-PAGE and antibodies against phospho-MEK, total MEK, phospho-ERK, and total ERK as described in Materials and Methods. The intensities of the phospho-MEK (B) and phospho-ERK (C) bands were quantified by using the software provided by the ChemiGenius imaging system and plotted as percentage of the respective phosphorylation intensities after 5 min of EGF stimulation and 0 min of chase. Intensities are adjusted for different loadings. Note that the values in C are obtained from a separate set of experiments with shorter chase times, an example of which is provided in Supplemental Figure S1. Control cells, square symbols; Tsg101 siRNA-treated cells, triangle symbols; and hVps24 siRNA-treated cells, round symbols. The error bars represent SEM of four experiments.