Goat anti rabbit serum

Protein samples were obtained from the testis and homogenized in sodium dodecyl sulfate (SDS) sample buffer supplemented with 1 mM phenylmethanesulfonyl fluoride. Samples were analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% (w/v) acrylamide gel with a 4% (w/v) acrylamide stacking gel using Laemmli buffer [16 (link)]. Each lane contained 10 μL gonad extract, and equal loading was confirmed by Western blot probed with an anti-β-actin (HuaAn Biotechnology Co., Ltd., Hangzhou, China) antibody. Samples were analyzed on 12.5% polyacrylamide gels under reducing conditions. After electrophoresis, the gels were electroblotted onto a polyvinylidene difluoride membrane. The membrane was treated in blocking solution (Roche, Shanghai, China) and incubated with primary antibody (1:100) for 2 h at room temperature. After washing with Tris-buffered saline (20 mM Tris-HCl, 0.9% NaCl, pH 7.4) containing 0.1% Tween-20 3 times at 10 min per wash, the membrane was incubated with goat anti-rabbit serum (1:5000) labeled with alkaliphosphatase (Bio-Rad) for 1 h at room temperature. After washing with Tris-buffered saline 3 times at 10 min per wash, detection was performed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche) as the substrate.

Pooled tubules of defined length (at least 100 mm/assay) were lysed in Tri-Reagent™ (Sigma) and protein was isolated as per the manufacturer's instructions (Chomczynski 1993 (link)). Samples were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide) and blotted onto nitrocellulose membrane as described (Shyjan and Levenson 1989 (link)). Blots were stained with Coomassie Brilliant Blue R-20 (Sigma) to verify protein transfer.
The blots were reacted with the following antibodies: rabbit anti-rat Na-K-ATPase α₁ (UPState Biotechnology, Lake Placid, NY) at a dilution of 1:3000; rabbit anti-Na-K-ATPase β₁ (UPState Biotechnology) at a dilution of 1:7000. The antibody–antigen complexes were visualized by incubation with goat anti-rabbit serum (dilution of 1:10,000; Bio Rad Laboratories), followed by enhanced chemiluminescence and autoradiography. Densitometry was performed using phosphor imager. Equal protein loading was verified using Coomassie Brilliant Blue R-20 staining.