Fig. 6. Relationship between extracellular calcium and tyrosine phosphorylation. (A) Spermatozoa were incubated in BWW -Ca²⁺ (lane 1), or BWW -Ca²⁺ supplemented with 50 (lane 2), 100 (lane 3), 300 (lane 4) or 600 (lane 5), µM CaCl₂. After 120 minutes incubation, 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 by 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 at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three similar experiments.