Figure 3: AKT activity impacts the levels of PHB phosphorylation (pT258) as well as the total and progressive motility of mouse mature sperm. (a) After wortmannin (a phosphatidylinositol 3-kinase inhibitor) treatment for 3 h, the phosphorylation levels of AKT (pS473; pT308) and PHB (pT258) in mouse cauda epididymal sperm were analyzed by western blotting. (b) Alpha-tubulin was used as a loading control and densitometric analysis used the relative ratios of AKT pS473 versus AKT, AKT pT308 versus AKT, or PHB pT258 versus PHB. Fifty micrograms of protein were loaded in each lane. (c) Vitality was evaluated via MitoTracker mitochondria Red CMXRos probe staining, and (d) the total and progressive sperm motility were determined by CASA. Mean ± s.e.m., and* P < 0.05;** P < 0.01; *** P < 0.001 determined by one-way ANOVA analysis with the Newman–Keuls posttest. PHB: prohibitin; AKT: protein kinase B; NS: not significant; CASA: computer-assisted sperm analysis; s.e.m.: standard error of mean; ANOVA: analysis of variance.