Western Blotting

Cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer. After heating at 99°C for 10 min to denature the protein, SDS-PAGE was performed to separate proteins, and a semidry transfer membrane was employed to transfer proteins onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 1 h at 37°C, and incubated at 4°C for 12 h with primary antibody. The next day, the membrane was incubated with the corresponding secondary antibody at room temperature for 1 h, and finally scanned using a Fluorescence & Chemiluminescence Gel Imaging System (Peiqing, Shanghai, China). Protein bands were quantified using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA).