Fig. 5.

Inhibition of Pyk2 reduces CR3-mediated phagocytosis of E. coli. a E. coli were incubated with PBS, IgG-depleted or heat-inactivated serum for 30 min at 37°C and analyzed for efficient opsonization via Western blot with an anti-C3 Ab (iC3b). b Serum-starved macrophages were activated with 200 ng/ml PMA and infected with FITC-labeled E. coli for 60 min (MOI 100) in the presence or absence of 10 µM PF431396. Cells were resuspendend and analyzed via flow cytometry. Uptake indices (% positive cells × mean fluorescence) were measured in the presence of trypan blue and are presented as relative values (normalized to mean of DMSO-treated control). Groups were compared by the Mann-Whitney U test. * p < 0.05. c Serum-starved RAW 264.7 cells seeded on poly-L-lysine-coated coverslips were activated with 200 ng/ml PMA and incubated for 30 min with FITC-labeled E. coli incubated with PBS, IgG-depleted or heat-inactivated serum. Cells were fixed and stained for Pyk2. Scale bars: 10 μm. d Normalized fluorescence intensity profiles of sites of E. coli infection over a 3-µm distance in samples of c. Intensities were normalized to maximal FITC fluorescence of bacteria and mean values of 7-10 intensity profiles are shown with error bars representing SEM.