EGF activates ribosomal S6 kinase, RSK1 to inhibit apoptosis in LY-treated LNCaP cells. (a and b) LNCaP cells were treated as in Figure 4a and b. Cells were harvested 1 h post-treatment and subjected to SDS–PAGE and immunoblotted with antiphospho RSK Ser380. (c) LNCaP cells were transfected with either control siRNA or siRNA specific for RSK1 or RSK2. At 48 h post-transfection cells were serum starved for 20 h before treatment with 10% serum-containing-medium (serum), LY in 10% serum containing medium (LY+serum), LY in serum-free medium (LY) and LY in serum-free medium and EGF (LY+EGF). Cells were harvested 1 h post-treatment and analysed by SDS–PAGE. Immunoblotting with anti-RSK1 or anti-RSK2 antibodies demonstrated specific silencing of RSK1 and RSK2, respectively. Apoptotic cell death at 24 h post-treatment was analysed by % of cells in sub-G1 phase (d) and caspase 3 activity (e). Results shown in (a–c) are from one representative experiment of at least three. (d) and (e) are a mean of three experiments performed in duplicates±s.e.