Knockdown of Ube2d proteins decreases ubiquitination of EGFR in HeLa cells.

HeLa cells were transfected with control siRNA (siNeg), or siRNAs targeting Ube2d1, Ube2d2, Ube2d3 and Ube2d4 separately or together (si2d) for 48 hours and then were either left untreated (0) or treated with 25 ng/mL EGF for 5 minutes (5). (A) RT-QPCR analysis of Ube2d mRNA expression as compared to ActinB expression in HeLa cells transfected with siNeg averaged for four independent transfections ± SEM. (B) RT-QPCR analysis of Ube2d knockdown efficiency in transfected HeLa cells. Numbers 1 through 4 on the X axis indicate the primers to Ube2d1 through Ube2d4, respectively, used in the PCR reaction. Average for three independent experiments ± SEM is shown. (C) Immunoblot analysis of whole cell lysates (WCL) using antibodies to detect EGFR, Cbl, all members of Ube2d family, and ActinB. EGFR was immunoprecipitated from the lysates and blotted for ubiquitin (Ub) or EGFR as indicated. (D) The fraction of ubiquitinated EGFR was calculated by densitometry analysis of EGFR IP from three independent experiments and plotted as an average ratio of Ub/EGFR ± SEM. Asterisk (*) denotes p < 0.05, (**) denotes p < 0.01. MW in kDa is shown to the left of the WB panels.