Ab231671

Germ cells, mouse tissues, and human spermatozoa were lysed in radioimmunoprecipitation assay buffer (P0013B, Beyotime, Shanghai, China), and protein concentration analysis was performed using a bicinchoninic acid (BCA) protein assay kit (23225, Invitrogen, Carlsbad, CA, USA). First, equal quantities of proteins were separated on 12% (w/v) SDS-PAGE and placed on a polyvinylidene fluoride (PVDF) membrane activated by methanol, followed by incubation with blocking solution (5% milk) at 37 °C for 1 h. The PVDF membranes were then incubated with antibodies against EMC10 (1:2000), ATP1B3 (1:5000; ab231671, Abcam, Cambridge, MA, USA), α-tubulin (1:2000; AF2827, Beyotime, Shanghai, China), and β-actin (1:10,000; ab6276, Abcam, Cambridge, MA, USA) at 4 °C overnight. EMC10 polyclonal antibody was generated as described previously [9 (link)]. After washing 3 times with 1× tris-buffered saline and Tween (TBST), the membranes were incubated with secondary antibodies against rabbit or mouse (1:1000; A0208 or A0216, Beyotime, Shanghai, China) at 37 °C for 1 h, prior to enhanced chemiluminescence detection (SB-WB012, Share-bio, Shanghai, China).

Spermatozoa were collected from the caput, corpus, and cauda epididymis of adult mice. Immunofluorescence was enhanced as previously described [36 (link)]. Briefly, sperm cells on slides were fixed with 4% PFA, incubated with 0.5% Triton X-100, and blocked with 10% goat serum (AR1009, Boster Biological Technology Co., Ltd., Wuhan, China). The slides were then incubated with antibody against ATP1B3 (1:100; ab231671, Abcam, Cambridge, MA, USA) at 4 °C overnight, and anti-rabbit Alexa Fluor 568 (1:1000; ab175471, Abcam, Cambridge, MA, USA) was used as the secondary antibody for incubation at 37 °C for 1 h. Peanut agglutinin (PNA) is a lectin that binds specifically to the outer acrosome membrane of sperm [37 (link)]. Here, FITC-labeled PNA (PNA-FITC; 1:2000, L7381, Sigma-Aldrich, St. Louis, MO, USA) was used to stain the sperm acrosome and co-incubated with secondary antibodies. Finally, sperm nuclei were revealed using 4′,6-diamidino-2-phenylindole (DAPI; 1:1000, D9542, Sigma-Aldrich, St. Louis, MO, USA) stain at 37 °C for 10 min. Images were acquired with confocal laser scanning microscopy (Leica, Heidelberg, Germany).

For immunohistochemical analysis, mouse testis and epididymis tissues were fixed in 4% (w/v) paraformaldehyde (PFA) at 4 °C overnight. Thereafter, tissues were dehydrated using a graded ethanol series, paraffin-embedded, and sectioned to approximately 5 μm. Next, the sections were de-waxed, rehydrated, subjected to antigen retrieval, and blocked with 10% donkey serum, followed by incubation with EMC10 (1:100) or ATP1B3 (1:100; ab231671, Abcam, Cambridge, MA, USA) antibody at 4 ℃ overnight. For colorimetric detection, the slices were thrice washed with 1× phosphate-buffered saline with Tween (PBST) and incubated with secondary antibody against rabbit (1:50; A0208, Beyotime, Shanghai, China) at 37 °C for 1 h. Subsequently, images were acquired under a BX-51 microscope (Olympus, Tokyo, Japan).