Western Blot Analysis of Protein Extracts

Proteins from frozen tissues and cells were extracted using RIPA buffer with 1% phosphatase inhibitor cocktail and PMSF. The lysates were collected following 30 min lysis on ice. Then the Pierce BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) were used to detected proteins concentration. The process of western blotting analysis was performed as described previously [12 (link)]. 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- onto the nitrocellulose (NC) membranes (PALL, CA, USA). The specific primary antibodies were used to incubate the membrane overnight at 4 °C. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody at 1:5000 dilution for 1 h at RT. The protein bands were observed with Enhanced Chemiluminescence (ECL) Detection Kit(Amersham, Piscataway, NJ, USA). Image J software was used to analyze the relative levels of proteins.