Fig. 5. Direct demonstration that Rab-GDI or monoclonal antibody to Rab4 release Rab4-GDP from early endocytic vesicles. Endocytic vesicles were preincubated with buffer or buffer plus 4 mM GDP or 4 mM GTP--S for 15 minutes at 37°C and subsequently incubated in the absence (A) or presence of Rab-GDI (B) or Rab4 monoclonal antibody (Rab4 mAb, C). After centrifugation, Rab4 was detected in supernatants or pellets by western blot as indicated. Rab-GDI caused Rab4 to appear in the supernatants and the amount of Rab4 was increased by preincubation of the vesicles with GDP and decreased by preincubation of the vesicles with GTP--S. Similarly, Rab4 mAb caused Rab4 to appear in the supernatants and the amount was increased by preincubation of the vesicles with GDP. The IgG band at 25 kDa results from crossreactivity of the anti-mouse secondary antibody with the Rab4 mAb that was incubated with the vesicles in the experiment.

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