Fig. 6. Effect from YART and YGT on the Rab5A distribution in HeLa cells.

HeLa cells were transiently transfected overnight with mCherry-C1-Rab5A (top; red) together with the indicated pEGFP-C1-toxin constructs (green). Afterward, cells were washed and incubated at 21°C for an additional 3 hours. Insets show in detail Rab5A-vesicle structure. Pictures are representative of three independent experiments. Scale bar, 10 μm. (A) Effects of YART^A or YART^A E160A on the distribution of overexpressed Rab5A. (B) Effects of active YART^A and YGT^G and inactive YART^A E160A and YGT^G AXA on the distribution of overexpressed mCherry-C1-Rab5A. (C) Quantification by MetaMorph imaging software of the size of mCherry-C1-Rab5A vesicles in the presence of the active (YART^A) or inactive ADP-ribosyltransferase domain (YART^A E160A). Cells of view are ≥20, and n = 3. Unpaired two-sample t test was used (***P < 0.001). (D) Quantification of (B) as described in (C). n.m., not measurable. (E) For analysis of the expression of pEGFP-C1-toxin constructs, HeLa cells were lysed after transfection and expression. Proteins were analyzed by SDS-PAGE, Western blotting, and immunostaining with anti-FLAG tag antibody. G indicates GFP-YGT^G and GFP-YGT^G AXA; A indicates GFP-YART^A and GFP-YART^A E160A.