Figure 2

Gαq and Gαi2 have opposing roles in chemotaxis. (A) Jurkat TAg T cells were transfected with G_αq-specific siRNA (GNAQ1103), G_αi2-specific siRNA (GNAI-2-1050) or control siRNA (GNAQ1103 M3). 48 hours post-transfection cells were placed in Costar Transwell wells with/without 10 ng/ml CXCL12. After 2 hours incubation the numbers of migrated cells were counted by flow cytometry Migration index was calculated as the number of cells migrating divided by the number of basal migrating mock tx cells (n = 8). (B) Cells were subjected to transfection as in (A). 48 hours post-transfection cells were stimulated with CXCL12 for the indicated time points. Cells were stained with anti-CXCR4 and surface expression of the receptor was analysed by flow cytometry (n = 3). (C) Cells were subjected to transfection as in (A). 48 hours post-transfection cells were stained with Fluo-4 and Fura Red AM and calcium fluxes were measured by flow cytometry. Grey lines indicates CXCL12 stimulated cells, black line unstimulated cells. Representative of 3 independent experiments. (D) Cells were subjected to transfection as in (A). 48 hours post-transfection cells were stimulated with CXCL12 for the indicated time points, lysed and subjected to immunoblot analysis with the indicated antibodies. Levels of immunoreactive protein were quantified by densitometric scanning from 3 independent experiments and normalized against total protein (Mean ± S.E.M.).