Figure 6: GTPase mislocalization at the leading edge by USP17 depletion.

HeLa cells were transfected with GFP-tagged Cdc42 (a) or RhoA (b) and either scrambled shRNA or USP17 shRNA1 as indicated. Cells were serum starved and either left unstimulated or stimulated with CXCL12 for 5 min. Cells were then fixed and stained for phalloidin-actin (red) and visualized by confocal microscopy. Scale bar, 10 μm. Arrows indicate areas of actin and GTPase plasma membrane colocalization. (c) HeLa cells were transfected with GFP-tagged RhoA along with either scrambled shRNA or USP17 shRNA1 and cells were serum starved for 12 h before imaging. At least ten brightfield and green TIRF images were captured per condition before and post stimulation with CXCL12 (100 ng ml⁻¹), in triplicate experiments. Fluorescence was scored on the basis of green TIRF per cell; values are mean±s.e.m.; and statistical analyses are relative to scrambled shRNA control by one-way analysis of variance. ***P>0.005. Grey bars indicate unstimulated cells, whereas black bars are CXCL12-stimulated cells.