Figure 4

ERβ1 induces ubiquitylation and degradation of EGFR. (A) Control (Lenti) and ERβ1-expressing MDA-MB-231 cells were incubated in the presence of 100 μM cycloheximide and 10 ng/ml EGF for the indicated times and analyzed for EGFR expression by immunoblotting. Treatment with EGF induces phosphorylation of EGFR and this accounts for the retarded electrophoretic mobility of EGFR at times 0.5 to 2. Lower panel: the graph represents the quantification of EGFR protein abundance from three independent experiments with SEM and P-value (*) ≤0.05% indicated. (B) Control (Lenti) and ERβ1-expressing MDA-MB-231 cells were incubated in absence or presence of 1 μM MG-132 for 6 h and analyzed for EGFR expression by immunoblotting. Lower panel: the bar graph represents the quantification of EGFR protein levels with SEM and P-value (*) ≤0.05% indicated. (C) Control (Lenti) and ERβ1-expressing MDA-MB-231 and Hs578T cells were incubated in the presence of 10 ng/ml EGF for 20 minutes. Lysates were immunoprecipitated with anti-EGFR antibody, followed by immunoblotting with the indicated antibodies. The bottom panel is the input control of cell lysates. (D) MDA-MB-231 cells were transiently transfected with control or ERβ siRNA (3#). 72 h after the transfection, cells were incubated with 10 ng/ml EGF for 20 minutes and analyzed as described in C. (E) Control (Lenti) and ERβ1-expressing MDA-MB-231 cells were serum starved, challenged with 10 ng/ml EGF for the indicated times and lysed under nondenaturing conditions. EGFR immunoprecipitates were probed with antibodies against EGFR and c-Cbl. The bottom panel is the input control of cell lysates.