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Fig. 1. EGFP-Rab7 colocalizes with monodansylcadaverine (MDC) and monodansyl pentane (MDH), markers for autophagic vesicles. CHO cells stably transfected with pEGFP-Rab7 wt, the mutants Q67L and T22N or with the vector alone (pEGFP) were incubated in full nutrient medium (j-k) or in EBSS medium (starved cells; a-i and m-o) at 37°C for 2 hours. Afterwards, cells were labeled with MDC (A) or MDH (B) and analyzed by fluorescence microscopy as described under Materials and Methods. Arrows indicate colocalization between Rab7 decorated vesicles and MDC. (C) Stably transfected CHO cells overexpressing GFP-Rab7T22N were incubated in control medium (full nutrient) or under starvation conditions for 2 hours. Protein extracts (membranes or cytosol) were subjected to electrophoresis on a 10% SDS-PAGE. The GFP protein was detected by western blotting (top panel) using specific antibodies. The bands were visualized with the ECL system (Amersham, NJ) and quantified (bottom panel) by densitometry. Figure represents one of three independent experiments.





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