Immunoblotting analysis.

Western blot analysis was carried out as previously described (32). Cells were lysed and subsequently sonicated in RIPA buffer (Shanghai Shenergy Biocolor BioScience & Technology, Shanghai, China) with PIC (Roche, Mannheim, Germany). Homogenates were centrifuged at 12,000 g for 10 min at 4°C, and protein concentrations of the supernatant were determined with the Pierce BCA Protein Assay Kit (catalog no. NCI3225CH; Thermo Fisher Scientific). Forty micrograms of protein for each sample, prepared in Laemmli sample buffer, was denatured in a boiling water bath for 10 min and then separated by SDS-PAGE, and the separated proteins were transferred to polyvinylidene fluoride membranes (GE Healthcare Life Sciences). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature, followed by incubation with primary antibody (anti-PRR antibody; catalog no. HPA003156; Millipore-Sigma) overnight at 4°C. After washing with TBST, membranes were incubated with secondary antibody (goat anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibody; catalog no. 31460; Thermo Fisher Scientific) for 1 h at room temperature and visualized with enhanced chemiluminescence (catalog no. 32106; Thermo Fisher Scientific). The densitometry of the bands was analyzed by using Image-Pro Plus 6.0. Exposure time was 3 min. The blot was stripped and reprobed with anti-β-actin antibody (catalog no. A1978; Millipore-Sigma). The expression of protein was calculated in relation to β-actin.