FIGURE 2.

PI3K activation by Syk is required for CX3CL1-induced Rho GTPase activation and chemotaxis. A, Ctrlsh and Syk shRNA-treated cells (Syksh) were stimulated with CX3CL1 for the indicated times, and the activation of Akt was assessed by Akt phosphorylation (p-Akt). Representative blots of p-Akt and total Akt are shown. p-Akt levels were quantified by densitometry and normalized to total Akt. Akt activity relative to Ctrlsh cells at time 0 was plotted (n = 3). Data represent mean ± S.E. *, p < 0.05 and **, p < 0.01 compared with Ctrlsh cells at time 0. B, RAW/LR5 cells were preincubated for 30 min with DMSO (vehicle) or 100 μm LY294002 prior to CX3CL1 stimulation for the indicated times. Representative blot of immunoprecipitated Syk (IP) probed for either phospho-specific Syk or Syk (upper panels). LY294002 efficacy was monitored by p-Akt levels in whole cell lysates (WCL; middle panels). Quantification of Syk activity relative to DMSO-treated cells at time 0 is shown (lower panels). C, BMMs pre-treated with DMSO (vehicle) or 100 μm LY294002 were incubated with CX3CL1 for the indicated times, and Cdc42 or Rac1 activity was determined at 15 sec (15″) or 1 min (1′), respectively, as described in the legend to Fig. 1 (n = 3). Mean ± S.E. *, p < 0.05 compared with DMSO-treated BMMs. D, RAW/LR5 cells were preincubated for 30 min with DMSO (vehicle) or 100 μm LY294002 before being subjected to a Transwell migration assay. CX3CL1-induced chemotaxis was expressed as fold induction compared with DMSO-treated cells in the absence of CX3CL1 (n = 3). Mean ± S.E. *, p < 0.05 compared with DMSO-treated cells.