Fig. 7. Relationship between ATP and tyrosine phosphorylation. (A) Human spermatozoa were incubated for 120 minutes in either complete BWW (open bars) or BWW -Ca²⁺ (closed bars). The four treatments consisted of the vehicle control BWW (lane 1), BWW-glucose (lane 2) BWW +2-deoxyglucose (lane 3) and BWW +1 µM rotenone (lane 4). 50 µl of cell suspension was taken for ATP measurements, as described in Materials and Methods. The graph represents the data from three experiments performed in quadruplicate; ^*P<0.05, ^**P<0.01. (B) Cells were incubated in either complete BWW (lanes 1-4) or BWW -Ca²⁺ (lanes 5-8) as shown. Following a 120-minute incubation, cells incubated in complete BWW (lanes 1, 5), BWW -glucose (lanes 2, 6), BWW +2-deoxyglucose (lanes 3, 7) or BWW +1 µM rotenone (lanes 4, 8) were harvested, 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. (C) 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 independent experiments.