Ho 1 ab189491

Quercitrin (standard product, HPLC purity ≥ 98%, product number: SQ8050, penicillin mixture (100×)) was purchased from Solarbio (Beijing, China). DMSO was purchased from Solarbio (Beijing, China). DMEM and fetal bovine serum were purchased from Gibco (Grand Island, NY, USA). fetal bovine serum (FBS) and trypsin EDTA solution were purchased from BI (Shanghai, China). RNAiso Plus was purchased from TaKaRa (Dalian, China); fluorescent quantitative from Genstar (Beijing, China). CCK8 and HiScript III RT SuperMix (R312-01) were purchased from Novozymes (Nanjing, China); the ROS kit and BCA protein concentration assay kit were purchased from Biyuntian Biotechnology (Shanghai, China). Acetylcysteine (NAC) was purchased from (Shanghai, China). The Ultrasensitive ECL chemiluminescence kit (P10300) was purchased from Xin Saimei Biotechnology (Suzhou, China). AcH3 (#9649), AcH4 (#8647), NF-kB p65 (#6956), Phospho-NF-kB p65 (#8214S), p38 MAPK (#8690), Phospho-p38 MAPK (#8203S), IκB-α (#8219S), Phospho-IκB-α (#8219S), AKT (#4691T), Phospho-AKT (#9271T), Anti-mouse IgG (#7076P2), Anti-rabbit IgG (#7074S), β-actin (#4970) and HRP secondary antibodies (#7076, 7074) were purchased from CST (Mass, USA). IL-8 was purchased from Proteintech (Wuhan, China). iNOS was purchased from NOVUS (MN, USA). HO-1 (ab189491) and NQO1 (ab80588) were purchased from Abcam (Cambridge, UK).

Protein expression was analyzed by western blot as described previously. Equal amounts of ovarian protein lysate from each group were added to the lanes of gels for electrophoresis and then transferred to a polyvinylidene fluoride membrane. Then, the membrane was blocked with 5% nonfat milk at room temperature for 1 h, eluted with PBST and incubated with primary antibodies against Nrf2 (16396-1-AP) (Proteintech), GCLC (12601-1-AP) (Proteintech), NQO1 (ab80588) (Abcam), HO-1 (ab189491) (Abcam), p16 (380,928) (Zhengneng Biotechnology) and klotho 382,164) (Zhengneng Biotechnology) at appropriate dilutions overnight at 4°C. The membranes were washed 3 times using PBST and then incubated in secondary antibody for 1 h. The membranes were detected by ECL and analyzed by Image Lab 4.0.1.