Expression, localization, and transactivation of wild-type and mutant Tax proteins in HeLa cells. HeLa cells were transfected with pCAGGS encoding Flag-tagged Tax proteins (TaxWT, TaxS240P, or TaxD247G) or the control pCAGGS without (A and B) or with the reporter plasmid pGV-BLTR and the reference plasmid pRL-SV40 ( C). Thereafter, 24 h after transfection, cells were subjected to western blotting ( A), confocal laser-scanning analysis ( B), and the firefly luciferase assay ( C). ( A) Western blotting was performed using an anti-Flag M2 MAb and anti-actin antibody as a control. ( B) For immunofluorescence, cells were fixed, permeabilized, and immunostained with anti-Flag M2 MAb followed by the Alexa-488-conjugated anti-mouse antibody (green). ( C) For the transactivation assay, cells were recovered and lysed, and the activities of firefly and Renilla luciferase were measured. For each sample, the firefly luciferase activity (pGV-BLTR) was normalized by reference to Renilla luciferase activity (pRL-SV40). Average values from triplicate transfections with standard deviations (error bars) are shown (*p < 0.01).