Fig. 4. Intracellular distribution of overexpressed PKC{alpha}, PKCßI, and PKCßII V5 domains and corresponding endogenous PKC isoforms. A–C, SK-N-BE(2) cells were transfected with vectors coding for fusion proteins between EGFP and V5 domains from PKC{alpha} (A), PKCßI (B), and PKCßII (C). Images demonstrate the localization of the V5-EGFP proteins visualized by the fluorescence of EGFP. D–F, immunofluorescence analysis of endogenous PKC{alpha} (D), PKCßI (E), and PKCßII (F) in SK-N-BE(2) cells. The immunoreactivity was visualized with TRITC-conjugated secondary antibodies. G—I, SK-N-BE(2) cells were transfected with vector coding for the EGFP-PKCßI V5 fusion protein, and 16 h after transfection, the cells were analyzed with immunofluorescence using an anti-PKCß antibody, which is not directed against the V5 region, as primary antibody. The same microscopic field depicts (G) the EGFP-PKCßI V5 protein visualized by the fluorescence of EGFP, (H) immunoreactivity of the anti-PKCß antibody visualized by a secondary TRITC-conjugated antibody, and (I) a phase-contrast image.