Sab5700632

Ovarian tissues were homogenized in RIPA buffer (Thermo Scientific, Rockford, IL, USA) and centrifuged at 12,000 × g for 20 min. The protein content of samples was determined using a BCA protein assay kit (Bio-Rad Laboratories, Carlsbad, CA). Aliquots of each sample were separated via 12% SDS-PAGE and then transferred onto a nitrocellulose membrane, which was blocked with non-fat milk for 1 h. The membranes were probed overnight at 4°C with antibodies against the following proteins: HO-1 (ab68477; 1:2000; Abcam); NRF2 (ab92946; 1:3000; Abcam), TNF-α (SAB5700627; 1:1000; Sigma), IL-1β (AB1413-I; 1:4000; Sigma), IL-6 (SAB5700632; 1:2000; Sigma); MVH (ab13840; 1:3000; Abcam); OCT4 (ab184665; 1:2000; Abcam); Ki67 (ab16667; 1:4000; Abcam), PCNA (mAb2586; 1:3000; Cell Signaling Technology), ATM (ab199726; 1:2000; Abcam), RAD51 (ab176458; 1:3000; Abcam), and GAPDH (ab181602; 1:3000; Abcam). After washing with PBST buffer solution three times, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Beyotime Institute of Biotechnology) for 1 h at 25 °C, and the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific Inc.). The blots were scanned and normalized to GAPDH for quantification.

Immunofluorescence was used to detect the positivity rate of selected molecules in each group of cells. The cells were fixed with 4% paraformaldehyde for 15 min, After the cell antigen was repaired and penetrated by Triton X-100 for 10 min, the cells were incubated with the following primary antibodies against: IL1B (AF7209; Beyotime China), FOS (ab222699; Abcam, Waltham, MA, USA), JUN (ab40766; Abcam), FCGR2A (HPA010776; Atlas Antibodies, Sweden), SRC (ab47505; Abcam), and IL6 (SAB5700632; Sigma-Aldrich, Germany) at 37 °C for 2 h. Primary antibody dilutions were set as follows: IL-1B, 1:100; FOS, 1:100; JUN,1:100; FCGR2A, 1:200; SRC, 1:100; IL6,1:200. PC12 cells were incubated with FITC-labeled secondary antibody (Invitrogen, Waltham, MA, USA) and H9c2 cells were incubated with Alexa-labeled fluorescent secondary antibody (Invitrogen) at 37 °C for 1 h. Secondary antibody dilutions were set as 1:200. Nuclei were stained with 0.5 µg/ml concentration of DAPI (Southern Biotech, Birmingham, Alabama, USA). The cells were imaged with a fluorescent microscope (Nikon ECLIPSE Ni-U/DS-Ri2, Nikon Co., Ltd. Kanagawa, Japan). The positive cell rate (five randomly selected high-power fields of view with at least 100 cells/group) was calculated as number of positive cells/total number of cells × 100.

Liver tissues were homogenized in lysis buffer containing phenylmethylsulfonyl fluoride in TissueLyser LT (QIAGEN, USA) homogenizer. The homogenate was centrifuged at 12.000 rpm for 20 min at 4°C. After total protein level adjustment, the samples were denaturated in loading buffer for 10 min at 95°C. Samples (40 μg of protein) were separated in 7.5% SDS-PAA gel and then transferred onto nitrocellulose membranes Immobilon® HAHY (Sigma-Aldrich, USA). Membranes are blocked with 5% fat-free dried milk for 2 hours at room temperature and incubated overnight at 4°C with rabbit anti-PPAR-γ antibody (SAB4502262, 1 : 1000, Sigma-Aldrich, USA), rabbit anti-pNF-κB p65 antibody (SAB4301496, 1 : 1000, Sigma-Aldrich, USA), rabbit anti-IL-6 antibody (SAB5700632, 1 : 1000, Sigma-Aldrich, USA), and rabbit anti-β-actin antibody (ZRB1312, Sigma-Aldrich, USA). After the washing, membranes were incubated with antirabbit IgG-Biotin (B8895), streptavidin, and HRP conjugate (18.152). The immunoreactive bands were stained using AEC Staining Kit (Sigma-Aldrich, USA).