Western blotting was performed according to the methods described previously by Wang et al. 15. Total protein was extracted from cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with phenylmethylsulfonyl fluoride (PMSF). After 30 min lysing on ice, the amount of total protein was determined using a bicinchoninic acid (BCA) kit. Equal amounts of protein in each sample were separated using 12% SDS-PAGE and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, USA). The membranes were then blocked with 5% skimmed milk at room temperature for 1 h, rinsed three times with TBST and incubated with the indicated primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibody for 2 h at room temperature. Antibody-antigen complexes were visualised using either a LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences) or a chemiluminescence imaging system (Clinx, Shanghai, China). Protein levels were semi-quantified using Image J 1.52 software.

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