BAD plays an important role in apoptosis mediated by LY in LNCaP cells. (a) LNCaP cells were left in 10% serum containing medium or serum starved for 20 h (0 h: serum and no serum, respectively) before being incubated with medium with 10% serum (Serum) for 1 h and analysed by SDS–PAGE and immunoblotting for phosphorylation of BAD at Ser118, Ser99 and Ser75. (b) LNCaP cells were serum starved for 20 h before being incubated with medium with 10% serum (Serum), serum-free medium (No serum) or serum-free medium with LY (LY). Cells were harvested at 30 min, 1, 3 and 6 h after treatment and analysed by SDS–PAGE and immunoblotting for phosphorylation of BAD at Ser75. (a and b) detection of total BAD (t-BAD) was used to ensure equal loading. (c and d) LNCaP cells were transfected with either BAD siRNA or control siRNA for 48 h followed by 20 h of serum withdrawal before treatment with 10% serum-containing medium (serum), serum-free medium and LY (LY) or serum-free medium and LY and EGF (LY+EGF). After 6, 12 and 24 h, cells were harvested for assessment of (c) percentage of cells in sub-G1 phase and (d) caspase 3 activity. (c) (insert) Western blot of lysates collected 72 h post-transfection showed effective BAD silencing. β-Actin showed equal loading. Result shown in (a and b) are from one representative experiment of at least three. (c and d) are a mean of three experiments performed in duplicates±s.e.