Figure 8.

ATD inhibits the recycling of TGF-βRI to the cell surface

(A) The serum-starved HFL1 cells were first treated with DMSO or ATD (1 μM) and chloroquine (Chlq) (100 μM) for 4 h and then performed the following experiments. The biotinylated protein was pulled down with streptavidin agarose, and quantification of the plasma membrane TGF-βRI was performed by immunoblotting. (B) Biotinylated serum-starved HFL1 cells were placed at 37°C for 30 min to allow for TGF-βRI endocytosis. The biotinylated protein was pulled down with streptavidin agarose, and quantification of the endocytosed TGF-βRI was performed by immunoblotting. (C) Then, the biotinylated HFL1 cells were placed at 37°C for 30 min to allow for TGF-βRI recycling. The quantification of biotinylated TGF-βRI included the internalized and recycled TGF-βRI that were subjected to streptavidin agarose pull-down and analyzed by immunoblotting. (D) The internalized TGF-βRI were subjected to streptavidin agarose pull-down and analyzed by immunoblotting. (E) Quantification of the percentage of internalized TGF-βRI. TGF-βRI internalization rate = total internalized TGF-βRI at 30 min/plasma membrane TGF-βRI × 100%. (F) Quantification of the percentage of recycled TGF-βRI. TGF-βRI recycling rate = (internalized and recycled TGF-βRI at 60 min – internalized TGF-βRI at 60 min)/total internalized TGF-βRI at 30 min × 100%). Three separate experiments were performed. The data are presented as the mean ± SEM. ^##P < 0.01, compared with control.