Serum and EGF do not prevent LY294002-mediated inhibition of AKT kinase phosphorylation and activity. LNCaP cells were serum-starved for 20 h before incubating with fresh growth media with 10% serum (serum), serum-free media (no serum), LY (25 μ M) in 10% serum-containing medium (LY+serum), LY in serum-free media (LY), EGF (100 ng/ml) in serum-free media (EGF), and LY and EGF in serum-free media (LY+EGF). After 1 h, cells were harvested and equal amount of protein was subjected to (a) SDS–PAGE and immunoblotting analysis to detect AKT phosphorylation at Ser473 and Thr308, and (b) AKT kinase activity assay. Endogenous AKT kinase of treated cells was pulled down using immobilized AKT 1G1 mouse monoclonal antibody and incubated with 1 μg of GSK-3 fusion protein in a kinase reaction buffer. Phosphorylation of GSK-3 was analysed by SDS–PAGE and immunoblotting. In (a) total AKT (t-AKT) was detected as control of equal loading. In (b) pull down AKT shows equal amount of the AKT kinase used for the assay. Results shown are from one representative experiment out of at least three.