FIG. 2.

Divergent effects of N-terminal and C-terminal Flag tag or YFP tag on CtBP cellular localization. (A to C) Endogenous CtBP1-L is mainly nuclear in Cos-1 and HeLa cells. Cos-1 and HeLa cells were either nontransfected (A) or transfected with the indicated expression vectors (B and C), and the expressed proteins were visualized by fluorescence microscopy either directly (B, yellow) or by using the indicated primary mouse antibodies and a fluorescein isothiocyanate-labeled secondary antibody (A and C, green). Cells were fixed with formaldehyde (A and C); living cells (B) were used directly for microscopic analysis. (D) Schematic representation of the fusion proteins used in this study. The constructs were generated as Flag-tagged (gray boxes), HA-tagged (gray boxes), and YFP fusion (yellow boxes) proteins as indicated. The unique N-terminal regions of CtBP1-L, CtBP2, and CtBP1-S are color coded as in Fig. 1A. Subcellular localization of each construct is summarized at right. C, predominantly cytoplasmic; N, predominantly nuclear; NC, nuclear and cytoplasmic.