Fig. 1. Impact of calcium on tyrosine phosphorylation in human spermatozoa. Spermatozoa (10x10⁶) were incubated in either BWW medium or in calcium-depleted medium (BWW -Ca²⁺) as indicated. (A) At the times indicated, the cells were centrifuged, washed and lysed in 2% SDS. 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were detected with western blot analysis using the anti-phosphotyrosine antibody, 4G10. The positions of the molecular mass markers (kDa) are shown on the left hand side. (B) The nitrocellulose membrane was stripped (2% SDS, 2 mM DTE, 50 mM Tris, pH 8.8) at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody, as a loading control. The western blot is representative of three independent experiments.