Polyvinylidene difuoride membrane

The distal corpus cavernosum was snipped and homogenized in radio immunoprecipitation assay (RIPA) lysis buffer, and then centrifuged at 12000g for 20 min at 4 °C. The supernatant was collected and protein concentration was determined by Coomassie brilliant blue G-250 working buffer. Equal amount protein was loaded to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis, the protein were then wet-transferred to polyvinylidene difuoride membrane (Merck Millipore) according to standard procedures. After blocking by 5% non-fat dry milk in TBS-t, the membranes were cut into pieces (referred to molecular weighs as preliminary experiment) and incubated with primary antibodies of anti-endothelial nitric oxide synthases (anti-eNOS) (1:1000, Abcam), anti-a-smooth muscle actin (anti-α-SMA) (1:1000, Abcam), and anti-androgen receptor (anti-AR) (1:1000, Abcam) for 24 h (at 4℃). The secondary antibody of β-Actin (1:200, Zen BioScience Co., Ltd. Chengdu, Sichuan Province, China) was incubated after membranes were washed. The densitometry of protein band was collected by Bio-Rad ChemiDoc MP (Bio-Rad, Berkeley, CA, USA) and its intensities were quantified by Image J sofware (National Institute of Health, Bethesda, MD, USA).

To explore the effects of Anxa7, Gtf2f1 and Mdk interference on related pathway in vitro, western blots were conducted on mice ovaries from the experimental and the control groups. We used RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) with protease inhibitor Phenylmethylsulfonyl fluoride (PMSF) to extract total protein from cells. Next, 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis were used to resolve 20 μg of the protein, and the bands were then electro-blotted onto polyvinylidene difuoride membranes (Millipore, Shanghai, China). Subsequently, the membranes were blocked with 5% skim milk for 1h and then incubated overnight at 4°C with the following primary antibodies. The primary antibodies used in the study were presented in Supplementary Table S1. After the incubation of the first antibody, the membranes were washed three times with Tris Buffered Saline Tween-20 (TBST). Following this, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5 000, Proteintech, Wuhan, China) or goat anti-mouse IgG (1:5 000, Proteintech, Wuhan, China). We then used enhanced chemiluminescence reagent to visualize signals according to the manufacturer’s protocol. The band intensity was quantified using Gapdh as internal control and measured with computer-assisted image system (Image Pro-Plus 6.0, Media Cybernetics, Bethesda, MD, USA).