Fig. 4.
The IκBα fragment enters the nucleus and retains NF-κB in the nucleus. (A) HeLa cells were transfected or not with a plasmid expressing the fusion protein FLAG-IκBα(WT)-myc or an IκBα fragment consisting of FLAG-IκBα-(1–249)-myc, which corresponds to the IκBα fragment generated by the viral protease. Transfected cells were washed with PBS, preincubated 90 min in serum-free medium with 100 μg/ml cyclohexamide (CHX), and then treated for 30 min with 10 ng/ml TNF-α plus 100 μg/ml CHX (CHX/TNF). The cells were then washed and subsequently incubated for 90 min in the absence of serum (Wash). Cells were fixed, permeabilized, and assayed for p65 (FITC, green) and IκBα (Cy3, red) by confocal microscopy (n = 3). (B) HeLa cells were transfected with a plasmid expressing 3Cpro and cotransfected with a plasmid expressing the fusion protein FLAG- IκBα(WT)-myc. Cells were treated as before and assayed for the FLAG-tagged amino-terminal IκBα fragment or the myc-tagged carboxyl-terminal IκBα fragment (red) and for p65 (green) (n = 3). (Magnification: ×60.)