Ab167139

For quantitative real-time PCR (RT-PCR) analysis, total RNA from cells was isolated using RNA simple Total RNA Kit (Tiangen Biotech), and miRNAs were performed using the miRcute miRNA isolation kit (Tiangen Biotech) according to the manufacturer’s instructions. RT-PCR was performed using Hairpin-it™ miRNAs RT-PCR Quantitation Kit (GenePharma, China) and samples were amplified for 40 cycles as follows: 95°C for 12 s, 62°C for 40 s, and 72°C for 30 s. The miR-106a expression was calculated relative to U6 snRNA. For Western blotting, proteins were loaded onto 12 or 15% SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (PVDF). Membranes were blocked in 5% non-fat milk in PBST for 1 h, and incubated with anti-ULK1(Abcam, ab167139), anti-ATG16L1 (Abcam, ab188642), anti-ATG7 (Abcam, ab52472), anti-LC3 (Abcam, ab51520), and anti-GAPDH (Abcam, ab245355). Immunoreactive band was performed using ECL reagent (Amersham Pharmacia) and quantified by using Image J software (NIH).

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.