Fig. 3.

Dynasore increases apical surface expression of downregulated in adenoma (DRA) via decreased endocytosis. A: overnight serum-starved Caco-2 cells were treated with 80 μM dynasore for 60 min and subjected to cell surface biotinylation. Biotinylated proteins were pulled down with NeutrAvidin-agarose beads, and surface and total fractions were resolved on a 7.5% SDS-polyacrylamide gel. Representative blot was probed with rabbit anti-DRA antibody. Biotinylated fraction represents surface DRA expression. Results are shown as ratio of surface to total DRA. Values are means ± SE of 3 different experiments. *P < 0.05 vs. control. B: apical surface of Caco-2 cell monolayers grown on Transwell filter inserts was treated with 80 μM dynasore for 60 min. Cells were fixed with paraformaldehyde and incubated with anti-DRA antibody (1:100 dilution) and then with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:100 dilution) and rhodamine-phalloidin (1:60 dilution) for 60 min. DRA is shown in green and actin in red. Orthogonal xz images were obtained with a Zeiss LSM 510 confocal microscope. C: overnight serum-starved Caco-2 monolayers were used for reverse cell surface biotinylation at 4°C and then incubated with 80 μM dynasore or vehicle (DMSO) at 37°C for 30 min. Excess biotin was cleaved by glutathione. Biotinylated proteins were pulled down with NeutrAvidin-agarose beads, and biotinylated and total fractions were resolved on a 7.5% SDS-polyacrylamide gel. Western blot was probed with rabbit anti-DRA antibody. Biotinylated fraction represents the endocytosed pool of DRA. Results are shown as ratio of endocytosed to total DRA. Values are means ± SE of 3 different experiments. *P < 0.05 vs. control.