Mouse anti beclin 1

Proteins from granulosa cells and follicles were collected by protein extraction kit and protein quantified by BCA (BestBio, Shanghai, China) method. First, the proteins were bathed in a metal bath at 95°C for 5 min. The lysate containing 20 μg total protein was separated by SDS-PAGE (Beyotime, Shanghai, China). The lysate was washed with PBS and transferred to PVDF membrane (Beyotime). After sealing with a blocking solution, the membrane was incubated overnight with the primary antibody at 4°C. The membrane was then washed in TBS for 3 times for 15 min each and incubated with antirabbit secondary antibody or antimouse secondary antibody at room temperature for 2 h. After washing with TBST, the protein was visualized by enhanced chemiluminescence (Beyotime). Density analysis of the bands relative to GAPDH protein was performed using ImageJ software. The following primary antibodies were used: rabbit anti-LC3B (cat. no. 3868T, Cell signaling Technology), mouse anti-Beclin1 (cat. no. 200675, ZenBio), rabbit anti-FMOD (cat. no. bs-12362R, Bioss), mouse anticaspase3(cat. no. bsm-33284M, Bioss), rabbit anticaspase8 (cat. no. Asp391, CST), and mouse anti-GAPDH (cat. no. bsm-33033M, Bioss).