EGF inactivates BAD and prevents cell apoptosis in LY-treated LNCaP cells. (a) LNCaP cells were serum-starved for 20 h before being incubated with serum-free medium (No serum), EGF in serum-free medium (EGF), LY in serum-free medium (LY), LY and EGF in serum-free medium (LY+EGF) or preincubated with U0126 for 3 h before being incubated with serum-free medium with LY, EGF and U0126 (LY+EGF+UO). Lysates were collected at 1, 3 and 6 h post-treatment for SDS–PAGE analysis and immunoblotted with antiphospho BAD Ser75 antibody. Membrane was stripped and reprobed with BAD antibody as loading control (t-BAD). (b) BAD protein was overexpressed in LNCaP cells by transfection with either BADwt plasmid encoding for murine BAD or control vector pcDNA3.1 as described in Materials and methods. At 48 h post-transfection cells were serum-starved for 20 h before being incubated in 10% serum-containing medium (serum), LY in serum-free medium (LY), LY and EGF in serum-free medium. Cells were harvested 1 h later for SDS–PAGE analysis and immunoblotted with antiphospho BAD Ser75 and antiphospho BAD Ser99 antibodies, which cross-reacts with both the human (23 kDa) and murine (29 kDa) phospho-BAD. β-Actin and total BAD (t-BAD) show equal loading. Results shown in (a and b) are from one representative experiment of at least three.