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Fig. 4. Effect of KN62 on the Ca2+-associated decrease in protein tyrosine phosphorylation. Immediately after selection using PercollTM 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.
