FIGURE 8.
The SH2-domain is required for Grb14 recruitment to CEACAM3 and inhibition of CEACAM3-mediated phagocytosis. A, 293 cells were cotransfected with GFP-tagged CEACAM3 or GFP-tagged CEACAM3ΔCT together with mKate2, mKate2-Grb14, or mKate2-Grb14ΔSH2 as indicated. 293 cells expressing the indicated constructs were infected with PacificBlue-labeled gonococci (total Ngo OpaCEA) at an MOI of 30 for 1 h. Cells were fixed and extracellular bacteria were stained with polyclonal antibody against gonococci and a Cy5-labeled secondary antibody (extracellular Ngo OpaCEA). Arrowheads indicate extracellular bacteria, small arrows point to internalized bacteria. Bars represent 10 μm. B, quantification of bacterial invasion from A. Boxes indicate the 25/75 percentiles and the median (black line) of the internalized bacteria per cell from three independent experiments. The 10/90 percentiles are indicated by the error bars. Statistical significance was evaluated by Wilcoxon's signed rank test (n = 60). n.s., not significant; **, p < 0.01; ***, p < 0.001.