Anti cleaved caspase 9

Cells were digested with pre-cooled RIPA lysate and protease inhibitors (Roche, Mannheim, BW, Germany) to extract total proteins and measure concentrations using the BCA method. SDS-PAGE was used to separate lanes containing 20 μg of proteins. Proteins were transferred to polyvinyl fluoride membranes (Millipore, Billerica, MA, USA) and then incubated in blocking buffer (5% skimmed milk) for 1 h. Membranes were then probed with anti-Bcl-2 (ProteinTech, 1:500), anti-Bax (ProteinTech, 1:500), anti-Cytc (Abcam, 1:500), anti-cleaved-caspase 9 (Abcam, 1:500) and anti-cleaved-caspase 3 at 4 °C overnight. The membranes were washed three times, each time for 10 minutes and then they were incubated in anti-rabbit IgG (Abcam, 1:5000) at room temperature for 45 minutes. The specific bands were revealed using the EC3 Imaging System (Analytik Jena US LLC, USA). Gel-Pro-Analyzer software was used to measure each band’s optical density.

Total protein was isolated from cell lysates by using RIPA buffer, and quantified by BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE and then transferred onto PVDF (Bio-Rad) membranes. After blocking with 5% skim milk in TBST for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). The primary antibodies used in this study were as follows: anti-p27 Kip1 (Abcam, Cambridge, MA, USA; 1:1000), anti-Bax (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000), anti-Bcl-2 (Abcam; 1:1000), anti-cleaved caspase 9 (Abcam; 1:1000), anti-CDK2 (Abcam; 1:1000), anti-Cyclin E1 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control.

Following treatment, whole cell lysates were acquired using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was quantified using a bicinchoninic acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) and 20 µg protein was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto nitrocellulose membranes and blocked in 5% non-fat milk for 2 h at room temperature. The membranes were then incubated with the following primary antibodies at 4˚C overnight: Anti-Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), anti-Bax (cat. no. ab32503; 1:1,000; Abcam), anti-cleaved caspase-9 (cat. no. ab2324; 1:1,000; Abcam), anti-cleaved caspase-3 (cat. no. ab32042; 1:1,000; Abcam), anti-caspase 3 (cat. no. ab13847; 1:1,000; Abcam), anti-caspase-9 (cat. no. ab32539; 1:1,000; Abcam) and anti-GAPDH (cat. no. ab8245; 1:1,000; Abcam). Following primary antibody incubation, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; 1:1,000; Cell Signaling Technology, Inc.) at room temperature for 2 h in the dark. Protein bands were visualized using an ECL Western Blotting Detection reagent (GE Healthcare) and densitometric analysis was performed using Image J software (v.1.52; National Institutes of Health).