CAR3 specifically regulates CHRN endocytosis. (A) C2C12 cells treated with AP (2 μg/ml), TFMS (2 mM) or AZA (1 mM) for 6 h, were lysed and subject to SDS-PAGE followed by immunoblot analysis using CAR3, CAR2 or ACTB antibodies. (B) C2C12 cells were treated with or without AP (2 μg/ml), TFMS (2 mM) or AZA (1 mM) for 6 h, followed by incubation with CHRN antibody (mAb210) at 4°C for 1 h, and then switched to 37°C for different times to induce CHRN endocytosis. After acidic washes, the cells were fixed and analyzed with flow cytometry. (C) C2C12 cells treated without or with AZA (1 mM), AP (2 μg/ml), TFMS (2 mM) for 6 h, and then labeled with biotin-CHRN antibody (mAb210). After further culture for 2 h, C2C12 cells were washed with acidic buffer, lysed and analyzed using SDS-PAGE and blotted with streptavidin-HRP. (D) The band densitometry was quantified using ImageJ software. Shown is a representative image of three experiments (A and C), and the quantitative data are presented as the mean ± SEM of 3 experiments (B). *p < 0.05, between the MβCD group and the control group; #p < 0.05, between the AP group and the control group; &p < 0.05, between the TFMS group and the control group.