Fig 1: S1P induces strong and sustained RhoA activation and brief, low-level Rac1 activation.(A-B) Analysis of RhoA and Rac1 activation by S1P and CCL19. Naïve CD4+ T cells were stimulated with S1P or CCL19 for the indicated times, then rapidly lysed and analyzed for GTP-bound RhoA and Rac1. Top panels show representative experiments; bottom panels show averages of multiple biological replicates (n=4 for RhoA; n=3 for Rac1). Fold activation was determined relative to the unstimulated controls. (C) Analysis of the Rac effector PAK1/2. T cells were stimulated with S1P or CCL19 for the indicated times, lysed with TritonX-100 lysis buffer, and analyzed by immunoblotting for phospho-PAK1/2, using GAPDH as a loading control. Top, representative Western Blot. Bottom, quantification from independent experiments (n=3). Fold increases were determined relative to the unstimulated controls. (D-E) Analysis of Rac1 activation in living cells. Purified human CD4+ T cells were transduced with Pak-PBD-mCherry, seeded onto fibronectin-coated glasses, and imaged before and after addition of 125nM S1P or 50mM CCL19 for up to 9min. Dark grayscale indicates Rac1-GTP levels. D, representative time-lapse images. E, Quantification of Rac1 activity at the membrane. Rac1-based protrusions were recorded, and their fluorescence intensity was compared to that of the same membrane area at steady state (dotted line). Plotted is the average Rac1 activity for S1P (blue) and CCL19 (red) over time. n = at least 20 separate cells for each condition, obtained from two separate experiments. For all panels, data represent means +/− StDev from multiple experiments. Statistical analysis was performed using an unpaired Student’s T test, relative to the unstimulated control. *, p<0.05; **, p<0.01; ***, p<0.005.