Hpa040343

Human spermatozoa from AY078 and control fertile groups were washed three times with PBS, dissolved using 1×SDS loading buffer (Beyotime Biotechnology,China), and denatured at 100°C to avoid protein loss due to inadequate lysis. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-SPEF2 (HPA040343, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-PLCζ1 (pab0367-P, Covalab, USA, 1:1000), rabbit polyclonal anti-SPAG6 (HPA038440, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACTL7A (HPA021624, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACROSIN (NBP2-14260, Novus Biologicals, Colorado, USA, 1:1000), rabbit polyclonal anti-RSPH1 (HPA017382, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-RSPH3 (17603-1-AP, Proteintech, Rosemont, IL, USA, 1:1000), and mouse polyclonal anti-β-actin (TA-09, ZSGB-Bio, China). After incubation with secondary antibodies at 37°C for 2 h, blots were visualized and analyzed(Tanon 5200,China).

IF was performed after samples had been pre-processed as described previously (32 (link)) using rabbit polyclonal anti-HYDIN (HPA067155, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-SPEF2 (HPA040343, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-PLCζ1 (pab0367-P, Covalab, USA, 1:100), rabbit polyclonal anti-ACTL7A (HPA021624, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-ACROSIN (NBP2-14260, Novus Biologicals, Colorado, USA, 1:200), rabbit polyclonal anti-RSPH1 (HPA017382, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-RSPH3 (17603-1-AP, Proteintech, Rosemont, IL, USA, 1:100), as well as mouse monoclonal anti-acetylated α-tubulin (T6793, Sigma, Castle Hill, NSW, Australia,1:500) antibodies and secondary anti-mouse Alexa Fluor 488 (Yeasen Biotechnology, USA, 34106ES60, 1:500) and anti-rabbit Alexa Fluor 594 antibodies (Jackson ImmunoResearch, USA, 111–585-003, 1:500). DNA was stained using Hoechst 33342 (Thermo Fisher Scientific, USA, 62,249, 1:1000).