Figure 6.

CAR3 suppresses endocytosis via a macroautophagy-mediated pathway. (A) Gastrocnemius from mice was homogenized in lysis buffer containing 1% NP-40, subject to SDS-PAGE and immunoblot analysis with the indicated antibody. Densitometric quantification of the indicated proteins over ACTB using ImageJ (right panel). (B) Gastrocnemius fixed, frozen sectioned, and permeabilized. After being blocked with 20% goat serum, the sections were incubated with anti-MAP1LC3A/B antibody and the appropriate Alexa Fluor 488-conjugated secondary antibody. After washing with PBS, the sections were examined with a confocal microscope and images were captured (left panel). Scale bar: 20 µm. MAP1LC3A/B-positive puncta were counted, and at least 50 cells were quantified. Puncta per cell are shown as mean ± SEM of 3 independent experiments (right panel). (C) C2C12 cells were transiently transfected with siScram or siAtg7 using Lipofectamine 3000. Forty-eight h later, the cells were either lysed and subjected to SDS-PAGE and immunoblot analysis with CHRN antibody (total CHRN, CHRN-t), or labeled with biotin-CHRN antibody (mAb210) and after 2 h the cells were washed with acidic buffer, lysed and analyzed by SDS-PAGE and immunblot analysis with streptavidin-HRP for endocytosed CHRN (CHRN-e). *p < 0.05, compared with the control group. Data are mean ± SEM of 3 independent experiments.