Atg12

The protein levels of the autophagy-related genes were determined by Western blot analysis. Briefly, 15 μg of total proteins extracted from the luteinized granulosa cells were electrophoresed on 10% or 12% SDS-PAGE gels and transferred onto PVDF membranes. After blocking, the membranes were washed with TBST and incubated with primary antibodies at 4 °C overnight. GAPDH (1:3000), Bcl-2 (1:1000), Bax (1:1000), P62 (1:1000) were purchased from Affinity Biosciences and Beclin1 (1:1000), Atg5 (1:1000), Lc3B (1:1000), Atg12 (1:1000), Atg16L (1:1000) were purchased from Cell Signaling Technology. The following day, the membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody at room temperature for 1 h. The bound antibody was visualized with an enhanced chemiluminescent detection kit (Engreen Biosystem, Beijing, China) and captured under a chemiluminescence imaging system (Tanon, Shanghai, China). The relative densities of the bands normalized by GAPDH, which was reported as a proper loading control than actin and tubulin were assessed by ImageJ software version 1.46r [61 (link)].

We carried out immunohistochemical analyses on 4% paraformaldehyde-fixed and paraffin-embedded testicular sections^{9 (link)}. We performed western blot analysis on testis extracts from wild-type, miR-471 Tg, sham control (Sham), flutamide-acyline (Flu+Acy), and androgen-replacement (Flu+Acy+T) groups; or cell extracts from miR-471 mimic-transfected 15P1 and TM4 Sertoli cells lines and primary Sertoli cells^{9 (link)}. We purchased antibodies to horseradish peroxidase-conjugated β-actin (1:50,000; Sigma-Aldrich, cat#A5316), β-tubulin (Sigma; T8328), DOCK180 (1:1000; Sigma-Aldrich; D9820), ACADL (1:1000; Sigma, #SAB2100020), TECPR1(1:1000; Abgent #AP7391b-ev), and Atg12 (1:1000; #A8731) from Sigma Inc. Antibodies for Sox9 (1:1000; Millipore, cat#AB5535), GAPDH(1:25,000; Sigma, Cat #G9295) and LC3B (1:3000; Sigma, L7543) were purchased from Millipore. Antibody to DOCK180 (1:1000; Thermo Scientific #MA5-15010) and Becn1 (1:1000; SC-11427) was purchased from Santa Cruz. Antibody to Ago2 was purchased from Wako Chemicals, Richmond, VA, USA (1:200; Cat #018-22021). RAB5 (1:1000; cat #2143), RAB7 (1:1000; cat#9367), Rubicon (1:1000; cat#8465), LC3A/B (1:2000; CST; cat #12741), cleaved caspase 3 (1:200; cat #9661) and Atg12 (1:1000; cat #418) antibodies were purchased form cell signaling technology. Antibody to Ago2 was purchased from.

We used the antibodies in parentheses to the following antigens: ATG9A (Abcam, catalog #108338), FLAG M2 (Sigma, F1804), PAK3 (Abnova, PAB2300), SPTLC2 (Abcam, ab23696), GFP-HRP (MACS, 130091833), actin-HRP (Sigma, A3854), beclin 1 (Cell Signaling, 3738), ATG7 (Cell Signaling, 8558), ATG5 (Cell Signaling, 12994), ATG12 (Cell Signaling, 4180), ATG16 (Cell Signaling, 8089), LC3 (Cell Signaling, 3868), LAMP-1 (Cell Signaling, 9091), SNAP29 (Abcam, 138500), gp41 (NIH AIDS Reagent Program, 2F5), TGN46 (Bio-Rad, AHP500G), anti-HIV immunoglobulin (NIH AIDS Reagent Program, HIV-Ig), Nef (NIH AIDS Reagent Program, 2949), α-tubulin (Sigma, T5168), VSV-G (Sigma, V5507), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen, A21206), Alexa Fluor 488- conjugated donkey anti-mouse IgG (Invitrogen, A21202), Alexa Fluor 555-conjugated donkey antirabbit IgG (Invitrogen, A31572), Alexa Fluor 555-conjugated donkey anti-mouse IgG (Invitrogen, A31570), Alexa Fluor 555-conjugated donkey anti-sheep IgG (Invitrogen, A21436), HRP-conjugated donkey anti-rabbit IgG (GE Healthcare, NA934V), and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, NXA931).

C2C12 cells were harvested in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Whole-cell protein extracts were quantified using the BCA assay, separated by SDS-PAGE 8–12%, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included Myogenin (Abcam, 1:1000, ab124800, Cambridge, MA, USA), MyoD (Santa Cruz, 1:200, sc-377460), HDAC9 (Abcam, 1:1000, ab59718), HIF1α (Abcam, 1:1000, ab179483), HIF2α (Abcam, 1:1000, ab179825), H3K9 (Abcam, 1:1000, ab32129), H3K14, H3K18, H4K16 (Cell Signaling, 1:1000), LC3I/II (Cell Signal, 1:1000, 12741), Beclin1 (Cell Signal, 1:1000, 3738), Atg5 (Cell Signal, 1:1000, 12994), Atg7 (Cell Signal, 1:1000, 8558), Atg12 (Cell Signal, 1:1000, 4180), p62 (Cell Signaling, 1:1000, 23214), p-GSK3β Ser9 (Cell Signal, 1:1000, 9323), GSK3β (Cell Signal, 1:1000, 12456), and active-β-catenin (Millipore, 1:800, 05–665). Stripped membranes were reprobed with GAPDH (Abcam, 1:4000, ab181602) as a loading control. Signal detection was performed using the ECL Kit (Beyotime Institute of Biotechnology) after incubation with an anti-rabbit or anti-mouse IgG secondary antibody (CoWin Bioscience Co., Beijing, China). Experiments were performed in triplicate.