Western Blot Analysis of Apoptosis and Signaling Pathways

The protein concentration was determined using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, United States) (Yi et al., 2016 (link)). Equal amounts of protein samples were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States) (Gu et al., 2015 (link)). The membranes were blocked in 5% nonfat dry milk for 2 h and incubated with primary antibody anti-Bcl-2 (1:1000; Abcam), anti-Bax (1:1000; Abcam), anti-caspase-3 (1:1000; Abcam), anti-cleaved caspase-3 (1:1000; Abcam), anti-phospho ERK1/2 (1:1000; Abcam), anti-ERK1/2 (1:1000; Abcam), anti-PI3K (1:1000; Abcam), anti-phospho PI3K (1:1000; Abcam), anti-phospho AKT (1:1000; Abcam), anti-AKT (1:1000;Abcam), and anti-GAPDH (1:5000; Abcam) overnight at 4°C. After three washes with Tris-buffered saline with 0.1% Tween-20 (TBST), HRP-conjugated secondary antibodies (Pierce) were used to incubate the membranes for 1 h at room temperature. The membranes were developed using a chemiluminescence reagent (Roche) after three times washed in TBST and exposed to Kodak exposure films. GAPDH served as an internal control in order to make normalization and relative quantitative analysis of target protein expression (Feng et al., 2016 (link)).