Serum-mediated phosphorylation of BAD is dependant on PI3K but not MEK activity. LNCaP cells were serum-starved for 20 h followed by incubation with serum-free medium (no serum), LY in serum-free medium (LY), medium with 10% serum (serum), LY in medium with 10% serum (LY+serum) or preincubated with U0126 for 3 h before being incubated with medium with 10% serum and U0126 (Serum+UO). Lysates were collected at 1, 3 and 6 h for SDS–PAGE analysis and immunoblotted with antiphospho BAD Ser75 antibody as in Figure 5a. Membrane was stripped and reprobed with BAD antibody as loading control (t-BAD). Results shown are from one representative experiment of at least three.