Fig. 4. Effect of KN62 on the Ca²⁺-associated decrease in protein tyrosine phosphorylation. Immediately after selection using Percoll^TM gradients, motile sperm were resuspended in medium not supplemented with CaCl₂, and 60 µM KN62 or KN04 were added. After 30 minutes, aliquots were supplemented with CaCl₂ (final concentration: 2.5 mM) and cells were incubated for 18 hours under capacitating conditions. Aliquots incubated in medium not supplemented with CaCl₂ (–Ca²⁺) and in the absence of the inhibitors (+Ca²⁺) or in the presence of 0.3% DMSO (DMSO) served as controls. Sperm protein extracts (2 x10⁶ cells per lane) were analysed by PAGE, immunoblotted and probed using a monoclonal antibody against phosphotyrosine (clone 4G10, Upstate Biotechnology). Molecular weight standards (x10^–3) are indicated on the left. Panel A, 2-minute exposure; Panel B, 10-minute exposure. A typical experiment is shown. This experiment was performed three times obtaining similar results.