Fig. 2. Intracellular localization of PI 3-kinase in human spermatozoa. (A) Western blot analysis of tail and head protein extracts from swim-up-selected spermatozoa separated onto 8% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was first blotted with anti-p85 PI 3-kinase regulatory subunit antibody (upper panel). Anti-ERK2 antibody (lower panel) was used to check for equal lane loading after stripping and reprobing of the same membrane. MCF7 cell lysate was used as positive control. Molecular mass markers are indicated on the right of the blot. Representative of two similar experiments. (B) Immunofluorescence analysis of fixed and permeabilized human spermatozoa probed with the antibody against the anti-p85 regulatory subunit of PI 3-kinase (b). Negative control avoiding the primary antibody is shown in a, whereas the respective fields observed in light transmission microscopy are reported in c and d. Arrows indicate sperm heads. Representative of four similar experiments.