Release of HB-EGF contributes to PAF-mediated EGFR transactivation. (A) SKOV-3 cells were treated with the HB-EGF neutralizing antibody (50 μg/ml) for 1 hour before stimulation with PAF (100 nM) or EGF (5 ng/ml) for 5 min, cells were lysed and lysates were evaluated by Western blotting. Data were normalized to total EGFR protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-EGFR compared to vehicle-treated cells. Representative blots for phosphor/total-EGFR are shown. (B) SKOV-3 cells were treated with PAF (100 nM) for the indicated time (30 min to 12 h) while measuring HB-EGF production. (C) SKOV-3 cells were serum starved and then pretreated with 20 μM GM6001 or HB-EGF neutralizing antibody (50 μg/ml) for 1 h. cells were then stimulated with 100 nM PAF for 2 h. The medium was harvested, and the amount of HB-EGF was measured. Bars represent the average of triplicates ± S.D.; * p < 0.05 indicate a statistically significant difference compared to the untreated control.