Figure 2

The ubiquitination or carboxyl-terminal region of Tat has little effect on its subcellular localization. (A) HeLa cells were cotransfected with GFP-Tat101 and His-Myc-tagged WT ubiquitin or the lysine mutants and treated with (+) or without (-) MG132. Cells were then stained with the DNA dye DAPI. The subcellular localization of GFP-Tat101 was examined by fluorescence microscopy. (B) Experiments were performed as in (A), and the percentage of nuclear GFP-Tat101 was quantified via ImageJ software. Bars represent the relative folds normalized to the untreated ubiquitin-K0 mutant transfection group. Experiments were done in duplicate and results were expressed as the mean ± SD. (C) HeLa cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone and treated with (+) or without (-) MG132. Cells were then subjected to nuclear staining with DAPI. The subcellular localization of GFP alone or GFP fusion proteins was analyzed by fluorescence microscopy. (D) Experiments were performed as in (C), and the percentage of nuclear GFP or GFP fusion proteins was quantified via ImageJ software. Bars represent the relative folds normalized to the untreated GFP vector transfection group. Experiments were done in duplicate and results were expressed as the mean ± SD.