Rac1 is downregulated by TSC1 without apparent changes in Rho activity.

<p>(A, B) Cells were subjected to the Rac1 (A) or RhoA (B) activity assay after culturing in normal (FCS+) or serum starvation conditions (FCS−). Western blot analysis was performed to detect Rac1 bound to PAK-1 PBD or rhotekin-PBD beads (top images) and in whole cell lysates (middle images). TSC1 expression was confirmed by immunoblotting of whole cell lysates (bottom images). Representative blots are shown. Quantifications of Rac1 (A) or RhoA (B) activity were performed using Image J (bottom). Levels of active Rac1 (A) or RhoA (B) were normalized to total Rac1 or RhoA, respectively, and expressed as a fold activation relative to CACL1-Hygro cells. Data are shown as means ± SE of three independent experiments. Significant differences were determined by one-way ANOVA with Dunnett’s post hoc comparison (**, p<0.01 vs. CACL1-Hygro).