Effects of HB-EGF and EGFR inhibitors on wound-induced EGFR phosphorylation. Growth factor-starved THCE cells were pretreated with 10 μg/mL HB-EGF neutralizing antibody, 0.5 μM AG1478, 50 μM GM6001, 10 μg/mL CRM197 (A) or 0 to 50 μM GM6001 (B) for 20 minutes and then wounded by comb-scratching, with unwounded (W −) serving as the control. Cells were lysed 15 minutes after wounding. For each reaction, 600 μg of protein was immunoprecipitated (IP) by 10 μg EGFR antibody and immunoblotted by mouse anti-PY99 (top), followed by reprobing with EGFR antibody (bottom). Data are representatives of three independent experiments. Left: molecular mass (kDa).