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Western blotting analysis

JH Jie Han
JY Jian Yang
QW Qiqi Wang
XY Xiang Yin
ZS Zewei Sun
CH Chaoyang Huang
GC Guoping Chen
LZ Liangrong Zheng
DJ Dongmei Jiang
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Differently-treated cells were harvested and lysed in radioimmunoprecipitation buffer (Beyotime Biotechnology, Shanghai, China). After the protein concentrations were determined, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h and incubated with primary antibodies overnight at 4 °C. Membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG. Protein bands were developed with the ECL Detection System (Life Technologies, Gaithersburg, MD, USA) and visualized with Image Lab Software (Bio-Rad, Hercules, CA, USA).

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