Western Blot Protein Detection Protocol

For western blotting, total protein samples were extracted by using a protein extraction buffer (1% IGEPAL, 150 mM NaCl, 50 mM Tris-HCl (pH 7.9), 10 mM NaF, 0.1 mM EDTA, and a protease inhibitor cocktail) as previously described [1 (link)]. The total proteins were separated followed by molecular weight by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The separated proteins were then transferred onto PVDF membranes (Millipore, Burlington, MA, USA), and the transferred membranes reacted with primary antibodies (1:1,000–1:5,000) and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) at 4 °C and room temperature for 12 h and 1 h, respectively. The Enhanced Chemiluminescence (ECL) Prime kit (GE Healthcare, Pittsburgh, PA, USA) was used for visualization of the differences of protein expression.