Western Blot Analysis of Protein Expression

Cells were lysed using a radioimmunoprecipitation assay buffer, containing protease and phosphatase inhibitors. Proteins (60 μg) were separated using 10% discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk for 1 h and then incubated with primary antibodies at 1:1000 dilution overnight at 4 °C. The membrane was washed in Tris-buffered saline containing 0.1% Tween 20 and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized by a densitometer using an enhanced chemiluminescence (ECL) detection kit (NCI4106; Thermo Fisher Scientific, Inc.) [37 (link)]. The same membrane was used for re-probing with an anti-GAPDH antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) at 1:1500 dilution. Band intensities were estimated using Image J, and the expression of proteins was normalized with respect to that of GAPDH.
Antibodies used have been listed in Table 1.