Ezrin, WASP, and Homer-3 localization in Jurkat cells stimulated with anti-CD3/CD28 antibody–coated beads. (A) Antibody-coated bead T-cell conjugates demonstrated redistribution of endogenous Homer-3 following T-cell receptor activation. F-actin accumulation (phalloidin-Alexa Fluor 594, red) in the contact area was induced by stimulation with anti-CD3/CD28 antibody–coated beads for 10 minutes, whereas no increase in actin accumulation was present at 2 minutes in Jurkat cells. Ezrin (green) and WASP (green) were recruited to the contact area along with actin accumulation after 10 minute stimulations. In contrast, Homer-3 (green) was recruited to the contact area after 2 minutes' stimulation, where increased actin accumulation was not observed. After actin accumulation in the contact area, Homer-3 was translocated into the nucleus (DAPI [4,6 diamidino-2-phenylindole], blue). Representative images showed that neither recruitment to the contact area nor nuclear translocation of Homer-3 was observed in the cells stimulated with anti-CD19 antibody–coated beads. (B) Jurkat cells were electroporated with Myc construct expressing coiled-coil domain (CCD) or EVH1 domain fragment of Homer-3. Twenty-four hours later, the cells were stimulated with anti-CD3/CD28 antibody-coated beads for 2 minutes and then stained with anti-Myc antibody. The CCD fragment of Homer-3 was recruited to the contact area, whereas the EVH1 domain fragment was not. Representative images were presented from experiments performed at least 3 times. Original magnification × 1000.