Figure 4.

Ephexin4^E295A induces RhoG-mediated processes to a greater extent than wild-type Ephexin4. (a–c) LR73 cells were transfected with the indicated plasmids and then incubated with TAMRA (5-Carboxytetramethylrhodamine, Succinimidyl Ester)-stained apoptotic thymocytes (a,c) or 2 µm carboxylate-modified beads (red fluorescence, (b)) for 2 h. Thereafter, cells were extensively washed with ice-cold phosphate-buffered saline (PBS) five times, trypsinized, and analyzed by flow cytometry. Cells positive for GFP and red fluorescence were considered to be phagocytes engulfing apoptotic cells (a,c) and carboxylate beads (b), respectively. n = 4. (d,e) LR73 cells were transfected with the indicated plasmids. One day later, cells were stained with Alexa Fluor 594-conjugated phalloidin and then with Hoechst 33342. Images were acquired by microscopy (d). At least 100 transfected cells were counted in randomly selected areas. Arrowheads indicate membrane ruffles. Scale bar, 20 µm. The percentage of GFP-positive cells with membrane ruffles was calculated (e). n = 3. Data are shown as the mean ± standard deviation and are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.