Fig. 6. Phosphorylation of CSK in capacitating murine spermatozoa. (A-D) Spermatozoa were pre-incubated with either 10 µM H89 (A,C) or the vehicle (B,D) for 5 minutes before the addition of 1 mM dbcAMP and 1 mM PTX to drive capacitation. After a further 40-minute incubation, cells were assessed for hyperactivated motility to confirm the attainment of a capacitated state in the vehicle controls. Capacitated and non-capacitated mouse spermatozoa were lysed and subjected to 2D PAGE as described in Materials and Methods. Proteins were then transferred to nitrocellulose membranes and, in the first instance, probed with anti-CSK antibody (C,D). Membranes were stripped and re-probed with the anti-phosphoserine antibody (A,B). Circles and arrows indicate the same position on all four images and indicate the serine phosphorylation of CSK in capacitated spermatozoa (B).