Rev erbβ

Protein lysates were prepared using CelLytic MT Cell Lysis Reagent (Sigma) as described previously^{14 (link)}, then subjected to SDS-PAGE, with the Precision Plus Protein Dual Color Standard marker (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). Primary antibodies used were REV-ERBα (mouse monoclonal, clone 4F6; 1:1000; Abgent), or REV-ERBβ (mouse monoclonal, clone D-8; 1:1000; Santa Cruz Biotechnology), or NLRP3 (mouse monoclonal, clone Cryo-2; 1:1000; Adipogen), or IL-1β (rabbit polyclonal, GTX74034; 1:1000; GeneTex), and incubated overnight at 4 °C. β-actin (mouse monoclonal, clone C4; 1:10,000; Millipore) was used as a loading control. Membranes were washed with tris-buffered saline with 0.1% Tween-20 (TBS-T), and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, 7076S, 1:5000; anti-rabbit, 7074S, 1:5000; Cell Signaling) in 3% skim milk in TBS-T. Proteins were visualized using Clarity Western ECL substrate (Bio-Rad), according to the manufacturer’s specifications. Densitometry quantification was performed using Image J 1.46 (NIH). All original blots are provided in Supplementary Fig. 5.

Fresh ovarian tissues were washed in PBS and homogenized. The homogenate was centrifuged at 3,000 × g for 10 min and the supernatant was collected for analysis. Granulosa and KGN cells were lysed with cell lysis buffer (Nanjing KeyGen Biotech, Nanjing, China). The cell lysate was used for analysis. Protein content in samples was measured using a bicinchoninic acid assay kit. Equal amounts of proteins were separated with SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, United States). PVDF membranes were blocked with non-fat milk (5%) for 2 h and then incubated with primary antibodies (REV-ERBα Santa-Cruz, sc-393215, 1:100; REV-ERBβ Santa-Cruz, sc-398252,1:100; PGC-1α Proteintech, 66369-1-Ig, 1:1,000; NRF1 Santa-Cruz, sc-28379, 1:200; TFAM Proteintech, 19998-1-AP, 1:1,000; LC3 Abcam, ab51520, 1:3,000; p62 Abcam, ab83134, 1:500; GAPDH Abcam, ab8245, 1:500) overnight at 4°C. Finally, membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody and immunoreactivity was detected by enhanced chemiluminescence.