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Fig. 1 | BMC Biology

Fig. 1

From: Phosphatidic acid-dependent localization and basal de-phosphorylation of RA-GEFs regulate lymphocyte trafficking

Fig. 1

Deficiency of RA-GEF in T cells impaired Rap1 activation and homing. a (Top) GTP-bound Rap1 was analyzed by a pull-down assay using GST-Ral-GDS-RBD. Wild-type (WT) or RA-GEF-deficient (DKO) T cells were stimulated with CCL21 at the indicated times. Bound Rap1 (Rap1-GTP) and total Rap1 were detected with anti-Rap1. (Bottom) Phosphorylation (p-) of Mst1/2 was examined with anti-phosphorylated Mst1/2. Total Mst1 is shown below. b (Top) WT and DKO T cells were stimulated with CCL21 for 10 min at 37 °C. The cells were stained with anti-LFA-1, alexaFluor 633 anti-Rat Ig, and FITC-CD44. DIC, differential interference contrast. Scale bar, 5 μm. Cells with segregated LFA-1 and CD44 accompanied with elongated cell shapes were considered to be polarized cells. (Bottom) The graph shows the percentages of the polarized cells (n = 50). *P < 0.001 versus WT cells. c (Left) Displacement and velocity of WT and DKO T cells were measured on ICAM-1 with or without CCL21 (n = 50). *1P < 0.001, *2P < 0.002 versus WT T cells. (Right) Representative tracks of WT or DKO T cells on the ICAM-1 in the presence of CCL21 are shown. Each line represents a single-cell track. d WT and DKO T cells were labeled with CFSE and CMTMR, respectively, and injected into normal mice. After 1 h, lymphocytes from injected mice were analyzed. (Left) Ratios of the number of DKO cells in the blood or lymph nodes relative to WT cells (adjusted to 1) (n = 3). *P < 0.001 versus corresponding cells. (Right) Representative flow cytometry profiles are shown. The numbers indicate the ratio of DKO to WT cells. e The numbers of T cells and B cells from the lymph nodes, spleen, and blood from WT or DKO mice are shown (n = 10). Cells were stained with anti-CD3, anti-B220, and anti-CD45 and analyzed using the flow cytometry. *P < 0.001 versus WT mice. Each bar graph represents the means ± SEM

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