Figure 6.

Effect of R-Ras knockdown on RalA activity on the endosomes. (A) Cos7 cells were transfected with expression vectors as indicated at the top of the panel. For knockdown, we used pSuper-R-Ras, an shRNA vector. For the rescue from knockdown, an R-Ras mutant resistant to the shRNA vector was expressed. Sixty hours after transfection, CFP and FRET images were obtained with a spinning confocal microscope. (B) MDCK cells were transfected with expression vectors as indicated at the top of the panel and imaged 20 h after transfection as in A. Outlined regions were enlarged and are shown in the insets. Arrowheads indicate representative vesicles. Bar, 10 μm. (C) Cells transfected with an empty pSuper vector and pSuper-R-Ras were selected as described in A, and the proteins were analyzed by immunoblotting with the antibodies shown on the left. (D) Histogram of the FRET level of Raichu-RalA on the endosomes. The histograms were drawn from the data obtained from 79 endosomes in four Cos7 cells, those obtained from 66 endosomes in six pSuper-R-Ras–expressing Cos7 cells and those obtained from 89 endosomes in nine Cos7 cells expressing both pSuper-R-Ras and pCXN2-5Myc-R-Ras-rRNAi. (E) HeLa cells were transfected with control siRNA or two different siRNAs for R-Ras. After 72 h, GTP-RalA levels in the cells were analyzed by Bos' pull-down method with GST-RalBP1-RBD. The knockdown of R-Ras was also confirmed by immunoblotting. (F) The histograms were drawn from the data obtained from 55 endosomes in two Raichu-RalA-expressing Cos7 cells transfected with siRNA for luciferase and those obtained from 83 endosomes in three Raichu-RalA–expressing Cos7 cells transfected with siRNA for Rgl2/Rlf.