Co-Immunoprecipitation Assay of PHB-AKT Interaction

The interaction of PHB with AKT was assessed using a coimmunoprecipitation (co-IP) assay with mouse caudal epididymal sperm after incubated in BWW for 3 h in a 5% CO2 incubator at 37°C. Briefly, collected sperm were homogenized in 1 ml of cold lysis buffer (150 mmol l⁻¹ NaCl, 200 mmol l⁻¹ Tris-HCl, 1 mmol l⁻¹ ethylene diamine tetraacetie acid [EDTA] pH 8.0, 1% Triton X-100, 0.5% NP-40, 1 × proteinase inhibitor cocktail, 1 × phosphatase inhibitor cocktail) for 40 min on ice before centrifugation (14 000g, 15 min, 4°C). Total protein concentrations of the lysates were quantified using the Bradford method (Pierce Biotechnology, Rockford, IL, USA). Lysate samples with 1 mg of total protein were gently rotated at 4°C overnight while incubated with 5 μg of PHB antibody (ab75766, Abcam, Cambridge, MA, USA) or 5 μg rabbit IgG. The PHB- or IgG-targeted immune-complex was collected after incubation with 50 μl of protein A+G agarose beads (Santa Cruz Biotechnology) via gently shaking at 4°C for 4 h before centrifugation at 1500g for 2 min at 4°C. The precipitate was washed three times with ice-cold washing buffer, resuspended in 4× protein loading buffer, and boiled for 10 min to dissociate the immune-complex from the beads. The supernatant was collected by centrifugation and subjected to standard SDS-PAGE and immunoblot procedures as previously reported.5 (link)6 (link)