Fig. 1. Identification of SRC and its binding partner PKA_c in murine spermatozoa. (A) Samples were taken from either the caput or caudal regions of the epididymis, lysed and run in a 10% SDS-PAGE. Western blot analysis was performed using the anti-SRC monoclonal antibody. A positive control of SRC (A431 cell lysates) was included to ensure the antibody cross-reacted with a protein of the appropriate size. Visualization was performed with standard ECL chemiluminescence. (B,C) Back-flushed murine spermatozoa were sonicated and Percoll-purified to obtain populations consisting of pure (>95%) (B) sperm heads or (C) sperm tails. (D) Approximately 2 µg of these fractions were then lysed and subjected to 10% SDS PAGE followed by western-blot analysis with anti-SRC antibody. (E) To demonstrate an association between SRC and PKA_c the above fractions were incubated with beads coated with anti-SRC antibodies and the precipitated proteins probed with an antibody against PKA_c. (F) The importance of sperm capacitation in this association between SRC and PKA_c was also confirmed in experiments involving the inmmunoprecitation of SRC-containing complexes from capacitated (incubated with 1 mM PTX and 1 mM dbcAMP for 45 minutes) and non-capacitated cells (freshly isolated from the cauda epididymis without incubation) followed by probing of these immunoprecipitates with anti-PKA_c antibodies. The western blot shows non-capacitated spermatozoa (lane 1) capacitated spermatozoa (lane 2) and a control incubation (lane 3), in which beads were incubated with sperm lysates in the absence of antibody. Arrows indicate the location of the IgG heavy chains (top) and IgG light chains (bottom).