Figure 7.

Fsk-Induced EGFR Phosphorylation and MAPK Activation Is Inhibited by AREG, EREG, and BTC Neutralizing Antibodies in Cultured Granulosa Cells

A, Inhibition of Fsk-induced EGFR phosphorylation by EGF-like growth factor neutralizing antibodies. Granulosa cells were serum starved before culture for 30 min in the presence of a combination of mouse neutralizing antibodies against AREG (3 μg/ml), EREG (2.5 μg/ml), and BTC (2 μg/ml) or identical concentration of mouse anti-IgG antibody as a control. Cells were next stimulated with Fsk (10 μm) or vehicle (DMSO) for an additional 30 min. EGFR was immunoprecipitated from granulosa cell extracts, and Western blot analyses were performed to detect phosphorylated and nonphosphorylated EGFR. Data shown are the mean ± sem of four separate experiments. *, P < 0.05. B, Inhibition of Fsk-induced MAPK activation by EGF-like growth factor neutralizing antibodies. Fsk-stimulated MAPK phosphorylation levels were examined by Western blot analyses in extracts of granulosa cells cultured for 30 min in the absence or presence of the three neutralizing antibodies against AREG, EREG, and BTC or mouse anti-IgG antibody as a control followed by 30 min stimulation with Fsk (10 μm) or vehicle (DMSO). Data are shown as the mean ± sem of five separate experiments. *, P < 0.05. Representative blots are shown for each experiment.