Fig. 2. Targeting of the mutant Rab7T22N to autophagic vacuoles is prevented by inhibitors of autophagosome formation. CHO cells expressing EGFP-Rab7T22N were incubated in EBSS medium (starved cells) at 37°C for 2 hours in the presence of 50 µM vinblastine (Vb), 0.1 µM bafilomycin A1 (BfA1), 200 nM wortmannin (WM) or with 30 mM 3-methyl adenine (3-MA). In this last case, cells were also preincubated with the drug for 2 hours before the starvation period. For the N-ethylmaleimide treatment (NEM) cells were pre-incubated for 15 minutes with 50 µM NEM before induction of starvation. Cells were washed and incubated for a further 2 hours in the starvation medium. Cells were subsequently labeled with MDC and immediately analyzed by fluorescence microscopy as described in Fig. 1.