Horseradish peroxidase hrp conjugated anti rabbit or anti mouse secondary antibodies

The specific procedures for WB have been described in a previous report 44 (link). Briefly, cells cultured with various treatments were collected, washed, and lysed with RIPA lysis buffer (Beyotime, Haimen, China). The acquired protein was quantified with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Next, equal amounts of protein were subjected to SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). Immunoblotting was performed with rabbit anti-NOX2 (Abcam, Cambridge, UK), rabbit anti-P65 (Cell Signaling Technology (CST), Danvers, MA), rabbit anti-p-P65 (CST), rabbit anti-IKBα (CST), rabbit anti-p-IKBα (CST), mouse anti-β-actin (Thermo Fisher Scientific), and mouse anti-GAPDH (Thermo Fisher Scientific) primary antibodies followed by horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific). Finally, the targeted proteins were visualized with ECL Plus WB detection reagents (Millipore).

The Puerarin and DDP were purchased from Shanghai Aladdin biochemical Technology Co., Ltd. DMSO was obtained from Beijing Solaibao Technology Co., Ltd. RPMI 1640 medium, fetal bovine serum and Trizol^TM kits were purchased from Gibco. The CCK-8 kits were obtained from Dojindo. RIPA lysate and ECL kit were purchased from Shanghai Yisheng Biotechnology Co., Ltd. The ELISA kits for human Wnt3a and VEGF proteins were purchased from Wuhan Huamei Biological Engineering Co., Ltd. and antibodies used in the Western blot experiments were bought from Santa Cruz. The primary anti-bodies and the horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies used here were purchased from Thermo (Shanghai, China). Other solvents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical or chromatographic grade.