Western Blot Protein Analysis

Cell lysates were separated through 7.5–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins separated on the gels were transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Hayward, CA, USA). The blots were blocked with 5% skim milk solution at room temperature for 1 h and then immunolabeled with primary antibodies (dilution 1:2000–1:10,000) overnight at 4 °C as previously described [53 (link)]. The membranes were washed using Tris-buffered saline with 1 × Tween-20 and then incubated with horseradish peroxidase-conjugated secondary antibodies (dilution 1:3000) at room temperature for 1 h. The immunolabeled protein bands were detected using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Band density was analyzed using ImageJ 1.5 software (NIH, Bethesda, MD, USA), and the expression levels were determined as the normalized ratio of each target protein to β-actin.