Figure 2

Subcellular localization of tagged p65/RelA and endogenous p65/RelA in activated Jurkat cells. (a) Jurkat cells did not show cleavage of p65/RelA in the cytosol or in the nucleus even after activation with PMA, as was determined by immunoblotting with an antibody against the carboxy terminus of p65/RelA. (b, c) Jurkat cells were transiently transfected with pCMV-p65wt-tag expression vector and then stimulated with PMA immediately after transfection. Analysis of protein expression was performed 18 hours after transfection by immunoblotting using an antibody against the carboxy-terminus of p65/RelA in the cytosol (b) or in the nucleus (c). Gel bands were quantified by densitometry and background noise was subtracted from the images. Relative ratio of optical density units was calculated regarding to the gel band with less optical density. (d) Analysis of subcellular distribution of tagged p65/RelA was also determined by confocal microscopy. Cells were transiently transfected with 1 μg of pCMV-p65wt-tag expression vector per million of cells and PMA or PHA was added immediately after transfection. After 18 hours, analysis of tagged protein expression was performed by confocal microscopy after staining with anti-FLAG tag M2 mAb and a secondary antibody conjugated with TexasRed. Two Jurkat cells from each experimental point related to two independent experiments are shown. (e) Analysis of the subcellular distribution of endogenous p65/RelA in Jurkat cells transiently transfected with 1 μg of pCMV-Tag1 control vector per million of cells and activated with PMA or PHA immediately after transfection. After 18 hours, analysis of p65/RelA distribution was performed by confocal microscopy after staining with an antibody against the carboxy-terminus of p65/RelA and a secondary antibody conjugated with Alexa 488. Two cells from each experimental point related to two independent experiments are shown.