Fig. 1. Presence and activity of PI 3-kinase in human spermatozoa. Immunoprecipitation of (A) p85 regulatory subunit and (B) p110 catalytic subunit of PI 3-kinase from human sperm lysates and of MCF-7 or PC3 cells, respectively, as positive controls. Immunobeads obtained with anti-p85 PI 3-kinase or anti-p110 PI 3-kinase antibodies (IP) were separated by 8% SDS-PAGE and the transferred proteins were revealed by western blotting with the same antibodies used for immunoprecipitation (IB). Molecular mass markers are indicated on the right (A) and on the left (B) of the blots. (C) PI 3-kinase activity in sperm samples treated or not with 10 µM LY294002 was evaluated by in vitro assay after immunoprecipitation with anti-p85 antibody. The spots correspond to PI 3-kinase catalytic product [³²P]phosphatidylinositol phosphate (PiP). Representative of four similar experiments. (D) Aliquot (1:4) of the p85 immunoprecipitates were subjected to western blot analysis with the same antibody, to ensure that protein G pulls down the same amount of the enzyme in both samples. Total lysates from PC3 cells and spermatozoa were run as positive controls.