Saposin C activates Akt signaling pathway in prostate cancer cells. A, cells were cultured up to 70% confluency in their complete media, serum-deprived for 24 h, and treated with 10% FBS or saposin C at 0.1, 1, or 10 nM for 10 min. A representative culture plate was also treated with LY294002 (LY; 50 μM) before treating with saposin C (at 1 nM for LNCaP and 10 nM for PC-3 and DU-145). Fifteen μg protein per sample was subjected to SDS-PAGE under reducing conditions and immunoblotting was carried out using phospho-specific Akt antibodies against serine 473 or threonine 308. B, non-radioactive in vitro kinase assay was performed to determine the effect of saposin C on Akt kinase activity as described in details in Materials and Methods. Briefly, cells were grown as described above and Akt was selectively immunoprecipitated from 250 μg protein using 20 μl of immobilized Akt 1G1 monoclonal antibody. Immunocomplexes were pelletted and resuspended in kinase buffer in the presence of 200 μM ATP and 1 μg of Akt/PKB substrate-glycogen synthase kinase fusion protein (GSK-3α/β) and incubated for 30 min at 30°C, allowing immunoprecipitated Akt (if activated) to phosphorylate GSK-3. After terminating the kinase reaction, phosphorylated GSK-3 was detected by SDS-PAGE and immunoblotting using phospho-GSK-3α/β antibody. Control loading was evaluated with anti-Akt antibody to determine total Akt-level. Each experiment was performed in duplicate, and the assays were repeated three times.