Quantitative Western Blotting Analysis

Western blotting was performed as previously described [40 (link)]. Briefly, the protein concentrations in cell lysates and tissue homogenates were measured using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts (20–30 μg) of protein were subjected to SDS-PAGE on 4%–12% Bolt^TM Mini Gels (Novex, Thermo Fisher Scientific, Waltham, MA, USA) and transferred to a Polyvinylidene fluoride (PVDF) membrane. Prestained protein ladders (GangNamstain^TM, iNtRON Biotechnology, Burlington, NJ, USA) covering a broad range of molecular weights were used to detect the molecular weights of the proteins. The membranes were blocked in 5% skim milk/bovine serum albumin (BSA) to reduce nonspecific binding and incubated with primary antibodies (1:1000 dilution) at 4 °C overnight. After reaction with a horseradish peroxidase-conjugated secondary antibody as appropriate, the proteins were detected using an enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. The x-ray films were scanned, and the optical densities of the bands were analyzed through densitometry using the computer-based Sigma Gel program, version 1.0 (SPSS Inc., Chicago, IL, USA). Density values were calculated in arbitrary units (AU) relative to the untreated control.