Fig. 6. The effect of guanine nucleotides on Rab4 immunolocalization on MT-bound endocytic vesicles. MT-bound Texas-Red–ASOR-containing early endocytic vesicles were incubated for 5 minutes in a motility chamber with buffer containing 4 mM GDP (top panels), or 4 mM GDP followed by an additional 5 minute incubation with 4 mM GTP--S (bottom panels). Monoclonal antibody against Rab4 was perfused into the chamber, incubated for 6 minutes, and visualized after addition of Cy2-labeled secondary antibody. Representative studies are shown in which Texas-Red–ASOR and rhodamine-labeled MTs are visualized in panels A and D. Rab 4 is immunolocalized in panels B and E. Merged images reveal that Rab4 is no longer immunolocalized to vesicles when pre-incubated with GDP (C). However, when GTP--S is perfused after GDP, Rab4 remains associated with these vesicles (F). These results are consistent with removal of Rab4-GDP but not Rab4-GTP from vesicles by the monoclonal antibody.