Fig. 5

PLD2 is critical for chemokine-dependent PA generation at the plasma membrane. a (Top) Displacement (left) and velocity (right) of T cells were measured on ICAM-1 with or without CCL21 in the presence or absence of 1 or 2 μM of CAY10593, CAY10594, and 5 or 10 μM of R59022 (n = 30). *¹P < 0.002, *²P < 0.001 versus WT T cells. (Bottom) Representative tracks of T cells treated with the indicated inhibitors with CCL21 are shown. Each line represents a single-cell track. b (Left upper) Displacement and velocity of scramble (control) or PLD2-knockdown cells were measured on the ICAM-1 with or without CXCL12 (n = 30). *P < 0.001 versus control cells. (Left lower) Representative tracks of control or PLD2 KD cells with CXCL12 are shown. Each line represents a single-cell track. (Right) BAF cells treated with or without CAY10594 were stimulated with CXCL12 at the indicated times, lysed, and subjected to the pull-down assay. Bound Rap1 (Rap1-GTP) and total Rap1 were detected with anti-Rap1. c PASS-GFP-expressing control cells were stimulated with CXCL12 for the indicated times. Time 0 represents the first time-lapse image; subsequent images were obtained in the same focal plane. Scale bar, 5 μm. d Distribution of PASS-GFP and RA-GEF-1 in BAF cells that were untreated (none) or treated with CXCL12 for 10 min in the presence (middle) or absence (top) of CAY10594 is shown. Scale bar, 5 μm. (Bottom) The graph shows the percentages of cells with the polarized membrane localization of PASS at the plasma membrane (n = 30). *P < 0.001 versus CXCL12-stimulated cells in the absence of CAY10594. Each bar graph represents the means ± SEM