The phosphorylation levels of PHB and the PI3K/AKT pathway in mouse sperm after treatment with wortmannin, and in human sperm with reduced motility from infertile males, were measured using western blotting analysis as previously described.5 (link)6 (link) The following primary antibodies were used: rabbit anti-PHB pT258 (1:500, GTX55299, GeneTex); rabbit anti-PI3 Kinase p85 (1:500, #4292), rabbit anti-AKT (1:500, #4060), rabbit anti-AKT pT308 (1:500, #9275) and rabbit anti-AKT pS473 (1:500, #9272) purchased from Cell Signaling Technology; rabbit anti-PI3K p85 alpha+gamma (Phospho Y199+Y467; 1:500, ab196001) and rabbit anti-PHB (1:500, ab75766) from Abcam; mouse anti-α-tubulin (1:1000, 66031-1-Ig) and mouse anti-β-actin (1:1000, 60008-1-Ig) from Proteintech Group, Inc. (Rosemont, IL, USA). To block the interference of IgG heavy and light chains on IP lysate blotting, a specific IP secondary antibody (the VeriBlot for IP secondary antibody [horseradish peroxidase, HRP; 1:40, ab131366]) purchased from Abcam was applied to detect the bound protein in the IP lysate. Images were captured using a Fujifilm LAS-4000 Mini Luminescent Imaging Analyzer (Fuji Film, Tokyo, Japan). The band intensities were quantified with ImageJ Software (National Institutes of Health, Bethesda, MA, USA).