Normal chemiluminescence detection

Western blot analysis was performed as previously described.^{32 (link)} Briefly, cells were rinsed twice with PBS and total proteins were solubilized in lysis buffer (150 mM sodium chloride; 50 mM Tris hydrochloride, pH 7.5;1% glycerol; 1% Non-idetp-40 substitute solution). Equal amounts of proteins were loaded and separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane, blocked by 5% skim milk, and probed with the specific (primary) antibodies, followed by treatment with secondary antibody conjugated to horseradish peroxidase (1 : 5000). Proteins were visualized by (enhanced) chemiluminescence detection (Pierce Biotechnology, Rockford, IL, USA) and exposure to X-ray film. β-Actin or Gapdh proteins were detected by normal chemiluminescence detection (Pierce Biotechnology) and exposed to less-sensitive X-ray film, which may result in a relatively weaker signal. All antibodies used in this study were obtained from Abnova (Taipei, Taiwan) or Abcam (Cambridge, MA, USA).

As previously described,^{29 (link)} cells were rinsed twice with PBS and solubilized in lysis buffer (150 mM sodium chloride; 50 mM Tris hydrochloride, pH 7.5; 1% glycerol; 1% Nonidet P-40 substitute solution (Sigma-Aldrich, Inc., St. Louis, MO, USA)). Equal amounts of total protein were loaded and separated by SDS-PAGE. Proteins were transferred to a polyvinylidenedifluoride membrane, which was blocked in 5% skimmed milk, and probed with specific (primary) antibody, followed by treatment with secondary antibody conjugated to horseradish peroxidase (1 : 5000). Proteins were visualized by (enhanced) chemiluminescence detection (Pierce Biotechnology) and exposure to X-ray film. Beta-actin or GAPDH proteins were visualized by normal chemiluminescence detection (Pierce Biotechnology) and exposed to less-sensitive X-ray film, which may result in a relatively weaker signal. All antibodies used in this study were obtained from Abnova (Taipei, Taiwan, China) or Abcam (Cambridge, MA, USA).