Figure 8.

CAR3 regulates endocytosis of CHRN through ER stress. (A) Gastrocnemius from mice was homogenized in lysis buffer containing 1% NP-40, subject to SDS-PAGE and immunoblot analysis with the indicated antibody. (B) Densitometric quantification of the indicated proteins over ACTB was performed using ImageJ. (C) C2C12 cells were transiently transfected with the indicated plasmids using Lipofectamine 3000. Forty-eight h later, the cells were treated with tunicamycin (TM; 2 μM) for 12 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. *p < 0.05, between the TM group and the control group; ^#p < 0.05, between the TM group and the Car3-TM group. (D) C2C12 cells were transiently transfected with the indicated specific siRNA using Lipofectamine 3000. Forty-eight h later, the cells with treated with PBA (2 μM) for 12 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. *p < 0.05, between siCar3 group and control group; ^#p < 0.05, between siCar3 group and siCar3-PBA group. (E) C2C12 cells were transiently transfected with siScramble or specific siRNA (siBag3) using Lipofectamine 3000. Forty-eight h later, the cells with treated with TFMS (2 mM) for 6 h followed by SDS-PAGE and immunoblot analysis with the indicated antibody. Data are mean ± SEM of 3 independent experiments (B, C, and D).