FIGURE 6.

GRKs differentially regulate signaling after activation of endogenous CXCR4 in HEK293 cells. A, HEK293 cells were loaded with the ratiometric calcium indicator Fura-2A/M 72 h after siRNA transfection. Cells were stimulated with 100 nm CXCL12, and changes in intracellular calcium were calculated from changes in fluorescence. Left panel, shown is a representative trace from six separate experiments. Right panel, shown is the mean ± S.E. increase in peak calcium transient calculated from six separate experiments. B, shown is the effect of GRK knockdown on CXCL12-mediated activation of ERK1/2. Seventy-two hours post-transfection cells were serum-starved for 6 h before stimulation with CXCL12 (100 nm). Shown is a representative Western blot (WB) from five independent experiments. C, left panel, pERK2 was normalized to total ERK2, and data are presented as the percent maximal ERK2 activation as compared with control (±S.E., n = 4). Right panel, comparison of maximal ERK2 activation (5 min) after stimulation with CXCL12 (100 nm) (±S.E., n = 4; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).