Immunohistochemical and Western Blot Analysis of Testicular Tissues

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.