Fig. 5. Tyrosine phosphorylation of sperm p110 kDa protein mediates LY294002 stimulatory effects on motility. (A) Effects of 1 hour treatment with LY294002 (LY, 10 µM) in the presence or absence of the tyrosine kinase inhibitor erbstatin (E, 12.5 or 25 µg/ml) on both forward and rapid motility of swim-up-selected spermatozoa. Sperm forward motility and rapid motility was evaluated by CASA. Data represent means±s.e.m. of five different experiments. #P<0.01 and ^*P<0.001 versus respective control, °P<0.001 versus respective LY; Student's t-test for paired data. (B) Western blot analysis of sperm lysates from swim-up-selected spermatozoa stimulated for 1 hour with 10 µM LY294002 and erbstatin (E, 12.5 or 25 µg/ml) together or alone. Equal amount (30 µg) of sperm proteins were subjected to 8% SDS-PAGE. Tyrosine phosphorylated proteins were revealed in chemiluminescence with PY20-HRP antibody. Molecular mass markers are indicated on the right of the blots. Arrowhead indicates the p110 kDa protein. Representative of five different experiments.