Pierce ecl western blotting substrate enhanced chemiluminescence system

Testes (40 pairs/sample) were dissected in Schneider’s media at room temperature within 30 minutes, the media was removed and the samples were frozen at -80°C until use. After thawing, testes were then lysed in 200uL of 2X Laemmli Sample Buffer + βME (BioRad, 161–0737). Samples were separated on a NuPAGE Tris-Acetate gel (3–8%, 1.5mm, Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore) using NuPAGE transfer buffer (Invitrogen) without added methanol. Membranes were blocked in 1X TBST (0.1% Tween-20) containing 5% nonfat milk, followed by incubation with primary antibodies diluted in 1X TBST containing 5% nonfat milk. Membranes were washed with 1X TBST, followed by incubation with secondary antibodies diluted in 1X TBST containing 5% nonfat milk. After washing with 1X TBST, detection was performed using the Pierce^® ECL Western Blotting Substrate enhanced chemiluminescence system (Thermo Scientific). Primary antibodies used were anti–α-tubulin (1:2,000; mouse, monoclonal, clone DM1a; Sigma-Aldrich) and anti-FLAG (1:2,500; mouse, monoclonal, M2, Sigma-Aldrich). The secondary antibody was horseradish peroxidase (HRP) conjugated anti-mouse IgG (1:10,000; Jackson ImmunoResearch Laboratories).

Testes (20 pairs per sample) were dissected and rinsed twice with 0.1 M phosphate buffer saline pH 7.2 (PBS), snap frozen and kept at −80 °C until use. Testes were homogenized in 100 µl (PBS), supplied with c0mplete protease inhibitor + ethylenediaminetetraacetic acid (Roche), and mixed with 100 µl of 2× Laemmli Sample Buffer (Bio-Rad). Cleared lysates were separated on a 12% Tris-glycine gel (Thermo Scientific) and transferred onto polyvinylidene fluoride membrane (Immobilon-P, Millipore). Mouse anti-α-Tubulin (clone 4.3; 1:3,000) (Walsh 1984) was obtained from the Developmental Studies Hybridoma Bank. The generation of polyclonal anti-Ste antibody (used at 1:10,000) was outsourced to Covance and was produced by immunizing guinea pigs with KLH-conjugated Ac-KPVIDSSSGLLYGDEKKWC (53–70 amino acids of Ste). Horseradish peroxidase-conjugated goat anti-mouse IgG (115-035-003; 1:10,000; Jackson ImmunoResearch Laboratories) and anti-guinea pig IgG (106-035-003; 1:10,000; Jackson ImmunoResearch Laboratories) secondary antibodies were used. The signals were detected by Pierce ECL Western Blotting Substrate enhanced chemiluminescence system (Thermo Scientific).