Quantitative Western Blot Analysis

Western blotting was performed as previously described [11 (link)]. The total protein was extracted from cells in the logarithmic growth phase using radioimmunoprecipitation assay lysis buffer containing protease and phosphatase inhibitors. Proteins (30 μg per group) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk at room temperature for 2 h, the nitrocellulose membrane was incubated with the primary antibody overnight at 4 °C. The primary antibodies are listed in Additional file 2: Table S2. After an incubation with a horseradish peroxidase-conjugated secondary IgG antibody (1:3000) at room temperature for 2 h, the nitrocellulose membrane was visualized using electrochemiluminescence (Bio-Rad GelDoc EZ), and the images were semi-quantitatively analyzed using ImageJ software. Each assay was performed in triplicate.