Western Blot Analysis of Protein Lysates

Tissue and cell lysates were prepared as described previously (Oh et al., 1997 (link); Shin et al., 2019 (link); Shrestha et al., 2018 (link)). Protein concentrations in the tissue and cell lysates were measured by Bradford assay and with Pierce BCA Protein Assay Reagent (Thermo Scientific, USA), respectively. Equal amounts of protein were resolved by SDS-PAGE and transferred to a PVDF membrane (Millipore). Blots were blocked with 5% skim milk or 3% bovine serum albumin in 0.1% Triton X-100 in PBS (0.1% PBS-T) for 50 min. The blots were incubated with primary antibodies for 1 h at room temperature. Then, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, USA) for 50 min and analyzed by enhanced chemiluminescence. α-Actin was used as a loading control.