FIG. 4.

Subcellular (confocal) localization of gE, gI, and gD in polarized epithelial cells. HEC-1A cells were coinfected with Ad(E1⁻)gE and Ad(E1⁻)gI (A to H) or infected with Ad(E1⁻)gD (I) for 48 h or were infected with HSV-1 for 8 h (J to L) or for 11 h (M to R). The cells were fixed with paraformaldehyde, permeabilized with 0.2% TX100, washed, and then incubated with various antibodies as follows. (A to C) Anti-β-catenin MAb (red) followed by Cy3-coupled anti-mouse antibodies and then with anti-gE MAb 3114 (green) coupled directly to Oregon Green followed by BODIPY-coupled anti-FITC antibodies (which react with Oregon Green); (D to G) rabbit anti-ZO-1 (red) and, simultaneously, anti-gE MAb 3114 (green) followed by FITC-coupled anti-mouse IgG antibodies and Texas Red-coupled anti-rabbit antibodies; (H) rabbit anti-ZO-1 (red) and, simultaneously, anti-gI MAb 3104 (green) followed by FITC-coupled antimouse IgG antibodies and Texas Red-coupled anti-rabbit antibodies; (I) rabbit anti-ZO-1 (red) and, simultaneously, anti-gD MAb DL6 followed by Texas Red-coupled anti-rabbit antibodies and FITC-coupled anti-mouse IgG antibodies; (J to O) as in A to C, anti-β-catenin MAb (red) followed by anti-gE MAb (green); (P to R) rabbit anti-β-catenin (red) and, simultaneously, anti-gD MAb DL6 followed by FITC-conjugated anti-mouse and Texas Red-coupled anti-rabbit antibodies. The left panels show HSV gE or gD in green, the same image with β-catenin or ZO-1 stained in red in the middle panels, and the two signals are combined in the right panels, except in panels G to I, which show combined signals of ZO-1 and either gE (G), gI (H), or gD (I). The arrows indicate borders of cells not in contact with other cells and without gE-gI and β-catenin.