Fig. 3

S1P activates Rap1 in RA-GEF-1/2-dependent manner, which is critical for chemotaxis toward S1P gradient. a (Top) Emigration of thymocytes toward CCL19 from the thymus lobes in Transwell chemotactic chambers. CD4 and CD8 profiles of CD3-gating cells that emigrated from WT and RA-GEF DKO thymus after 3 h of incubation with CCL19 are shown. (Middle) The numbers of emigrated cells from three independent experiments using WT or DKO mice. *P < 0.001 versus corresponding WT cells. (Bottom) Thymus sections from WT and DKO mice stained for CD4 (green) and CD8 (red). Scale bar, 200 μm. b The migration of thymocytes toward S1P in Transwell chemotactic chambers. The cell numbers of CD4⁺ or CD8⁺, and CD62L^high cells that migrated toward S1P after 3 h of incubation with S1P (n = 3). *P < 0.001 versus corresponding WT cells. c (Top) WT or DKO T cells were stimulated with 1 μM of S1P for the indicated times, lysed, and subjected to a pull-down assay. Bound Rap1 (Rap1-GTP) and total Rap1 were detected with anti-Rap1. (Bottom) Phosphorylation (p-) of Mst1/2 in WT or DKO T cells stimulated with 1 μM of S1P for the indicated times was examined with anti-phosphorylated Mst1/2. Total Mst1 is shown below. d (Top left) The experimental set-up of scheme. Time-lapse sequences of WT and DKO T cells migrating toward the S1P source were recorded. (Top right) Displacement of WT and DKO T cells (n = 30). *P < 0.001 versus WT T cells. (Bottom) Representative tracks of WT or DKO T cell on ICAM-1 in response to S1P gradient are shown. Each line represents a single-cell track. Each bar graph represents the means ± SEM