Ab28172

Fixed tissues were dehydrated using a full-automatic dehydrator, paraffin embedded and then sectioned into 5-mm thick samples. First, the dewaxed sections were placed into the dyeing tank with 3% methanol hydrogen peroxide at room temperature for 10 min. The samples were rinsed with PBS three times for 5 min each. The slices were dipped into 0.01 M citrate buffer (pH 6.0) and then heated in the microwave until boiling for 5 min interval; the heating was repeated once more. After cooling, the slice was washed with PBS two times for 5 min each. The sections were then blocked with a blocking serum (ZLI-9021, ZSGB-BIO) at room temperature for 20 min. The sections were incubated with the AR (C-19) antibody (1:100; sc-815, Santa Cruz) at 4°C overnight and then with a secondary antibody (1:250; sp-9001, ZSGB-BIO) for 30 min at 37°C. Samples were rinsed with PBS three times for 5 min each and then processed with the Concentrated DAB kit (K135925C, ZSGB-BIO). Sections were then dehydrated in alcohol, cleared in xylene and mounted in synthetic resin. Mitochondria staining of paraffin tissue sections was carried out using the anti-prohibitin antibody mitochondrial marker (1:100; ab28172, Abcam). The mean density of immunohistochemical staining was measured using Image-Pro Plus (IPP). n = 4 per group; five technical replicates.

The mitochondrial fractions were treated with a sample buffer (50 mM Tris, pH 6.8, 10% glycerol, 1.3% SDS, and 100 µM DTT) and heated at 90 °C for 5 min. The samples were loaded in a 12% SDS-polyacrylamide gel, separated by electrophoresis, and then transferred to a PVDF membrane. After 1 h of incubation with a blocking buffer (PBS, 3% Tween, and 3% albumin), the membrane was washed and incubated overnight with the primary antibodies at 4 °C. The primary antibodies used were rabbit monoclonal anti-AQP8 antibody [EPR8397] (ab133667) (Abcam, Cambridge, UK) (1 μg/mL), ornithine transcarbamylase (AV41766) (OTC) (1 μg/mL), and carbamoyl phosphate synthetase 1 (AV45689) (CPS1) (0.5 μg/mL) (Sigma-Aldrich). Rabbit antibody (ab28172) (Abcam) against prohibitin (0.5 μg/mL), an inner mitochondrial membrane protein, was also used. After that, the blots were repeatedly washed and incubated with a secondary antibody against rabbit IgG conjugated to horseradish peroxidase for 1 h. Finally, proteins were detected using a chemiluminescent kit (ECL Pierce, Thermo Fisher Scientific) and via the exposure of autoradiographic films (Cytiva), and the protein bands were analyzed based on densitometry in Fiji (ImageJ) v. 2.9.0/1.53t.

The total proteins in the testis tissues were separated using a Total Protein Extraction Kit (BC3711, Solarbio). The protein concentration was measured using a Protein Assay kit (Beyotime). Next, the protein samples were separated by 10% SDS-PAGE and electrically transferred to PVDF membranes (Millipore, MA, USA). After sealing with 3% bovine serum albumin at room temperature for 1 h, the membranes were hatched with the primary antibodies (rabbit anti-prohibitin (PHB), ab28172, 1:1000; rabbit anti-Beclin-1, ab62557, 1:2000; rabbit anti-LC3II/I, ab128025, 1:1,000; rabbit anti-p62, ab56416, 1:1,000; rabbit anti-SCF, ab64677, 1:1000; rabbit anti-Parkin, ab15494, 1:1,000; rabbit anti-LKB1, ab199970, 1:1,000; rabbit anti-AMPKα, ab131512, 1:500; rabbit anti-phosphorylated AMPKα (p-AMPKα), ab133448, 1:10000; rabbit anti-ULK1, ab167139, 1:1000; rabbit anti-p-ULK1, ab203207, 1:1000; rabbit anti-β-actin, ab8227, 1:5000; Abcam, Cambridge, UK) overnight at 4 °C. The membranes were incubated with goat-anti-rabbit IgG (H + L)-HRP (1:10000, ab6721, Abcam) for 1 h at room temperature and then rinsed thrice with TBST thrice. Protein bands were visualized using an Electrochemilluminescence (ECL) chemiluminescence kit (WBULS0500; EMD Millipore), and band intensity was analyzed with Image-Pro Plus 6.0.