Fig. 2. Identification of the phosphorylated forms of SRC. (A-D) Spermatozoa were pre-incubated for 5 minutes with either 10 µM H89 (B,D, non-capacitated cells) or the vehicle (A,C; capacitated cells) before the addition of pharmacological agents to drive sperm capacitation (1 mM dbcAMP and 1 mM PTX). After a further 40-minute incubation, the cells were assessed for hyperactivated motility. Populations containing at least 95% hyperactivated motility (A,C) or less than 5% hyperactivated motility (B,D) were lysed and subjected to 2D PAGE as described in Materials and Methods. The proteins were then transferred to nitrocellulose membranes and probed with anti-pY416 antibody. Arrows in A and B indicate the position of SRC. The membranes were then stripped and re-probed with anti-SRC as a positive loading control (C,D). Arrows in C and D indicate the position of SRC and its isoform phosphorylated at Y416. The encircled spots were also present when the membrane was probed with secondary antibody alone, indicating that these signals were the result of non-specific interactions.