Figure 7.

CD treatment blocks the recycling of Tac from the tubular compartment back out to the PM. Anti-Tac antibodies bound to the surface of cells expressing ARF6 and Tac were loaded into the tubular compartment by incubation with 1 μM CD at 37°C for 30 min. The cells were chilled to 4°C, rinsed with low pH buffer to remove the remaining surface anti-Tac antibodies, and then either fixed immediately (Load) and assessed for the Tac antibody loaded, or warmed to 37°C for 30 min in the absence or presence of CD before fixation. Tac antibody that reappeared on the cell surface was detected by incubation with fluorescently labeled secondary antibodies in the absence of detergent permeabilization (Surface Reappearance); ARF6 was subsequently localized in these cells after permeabilization.