Fig. 3. Ran is required for JNK1/2 and p38α/β nuclear translocation. HeLa cells were grown on cover slides to 30% confluence. SiRNA of Ran and a scrambled siRNA (SiScr; serves as a negative control) were then transfected to the cells using Dharmafect according to the manufacturer's instructions. Forty-eight hr after transfection, the cells were serum starved, and then either stimulated with anisomycin (anis; 0.5µg/ml, 15 min), or left untreated (NT) as indicated. The cells were then fixed, stained using the indicated anti MAPK Abs and visualized using a fluorescent microscope. The bar in the upper right panel is of 20 µM. B. SiRNA reduces the amount of Ran. To confirm the efficiency of the siRNA, cell were transfected with siRNAs as described, and were subjected to a Western blot analysis with the indicated Abs (lower panel). C. Quanntification. The results in A were quantified (total of 60 cells in 4 fields from two distinct experiment. The fold change in cells with nuclear staining (>40%) upon anisomycin stimulation was calculated in cells treated with anisomycin in both control Si Scr and Si Ran. * - P<0.01 between Anis treated and untreated cells.