Goat anti rabbit igg hrp antibody

The cells were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) on ice for 20 mins. Protein concentration was determined using a BCA Detection Kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred to nitrocellulose membranes (Schleicher and Schuell, Germany). The membranes were incubated in 5% skim milk for 1 h at room temperature and washed three times on a shaker in Tris-buffered saline containing 20% Tween-20 (TBST). Then, the membranes were incubated with antibodies against BAX (Cell Signaling Technology, Danvers, MA, USA), Rabbit monoclonal anti-CDK6 (cat. no. ab124821; 1:50,000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-GADPH (cat. no. HRP-60004; 1:10,000; Proteintech group, lnc; Manchester, UK) and β-tubulin rabbit Polyclonal Antibody (cat. no. AF0001; 1:1000; Beyotime Biotechnology, Shanghai, China). Goat anti-mouse IgG-HRP antibody (cat. no. ab A0216; 1:1000; Beyotime Biotechnology, Shanghai, China) and goat anti-rabbit IgG-HRP antibody (cat. no. ab A0208; 1:1000; Beyotime Biotechnology, Shanghai, China) were used as secondary antibodies. After application of the secondary antibody, the blots were visualized using the electrochemiluminescence (ECL) system (Bio-Rad Laboratories, Hercules, CA, USA).

Triptolide was purchased from Chengdu Biopurify Phytochemicals Ltd. Cholesterol (Chol), distearoyl phosphatidylglycerole (DSPG) and egg yolk lecithin (PC-98T) were sourced from AVT (Shanghai) Pharmaceutical Tech Co., Ltd., Shanghai, China. GSH and GSSG assay kit, Annexin V-FITC/Propidium iodide (PI) Apoptosis Detection Kit, Cell Cycle and Apoptosis Analysis Kit, Bicinchoninic Acid (BCA) Protein Assay Kit and goat anti-rabbit IgG/HRP antibody were obtained from Beyotime Institute of Biotechnology, Nanjing, China. Reactive Oxygen Species Assay Kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. RPMI 1640 medium, trypsin, and fetal bovine serum (FBS) were provided by Gibco BRL, USA. Anti-caspase-3 antibody and anti-PARP-1 antibody were soured from Cell Signaling Technology, USA. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit monoclonal antibody was purchased from WuXi AppTec, Shanghai, China.