FIGURE 6.
Knock-down of Grb14 increases CEACAM3-mediated phagocytosis. A, Grb14 expression in HeLa-CEACAM3 cells and HeLa-CEACAM3 cells transduced with empty lentiviral particles (empty vector) or with lentiviral particles encoding a Grb14-directed shRNA (shRNA-Grb14) was analyzed by Western blotting using polyclonal anti-Grb14 antibody (top panel). Expression of CEACAM3 was detected with monoclonal anti-CEACAM antibody (middle panel). After stripping of the membrane, equal protein content of all samples was verified using an anti-tubulin antibody (lower panel). B, HeLa cells, untransduced HeLa-CEACAM3 cells (CEACAM3), and HeLa-CEACAM3 cells transduced with the empty vector or with shRNA-Grb14 were infected with fluorescein-labeled OpaCEA protein-expressing N. gonorrhoeae (MOI 20). After 60 min, bacterial uptake was measured by flow cytometry. Fluorescence of extracellular, cell-associated bacteria was quenched by addition of trypan blue. Bars represent mean values ± S.D. of four separate experiments. n.s., not significant; *, p < 0.05; **, p < 0.01.