Figure 4. NF-κB p65 translocates to the nucleus normally in anergic cells.

(A) Purified T lymphocytes were stimulated for the indicated time points and cytoplasmic and nuclear fractions were collected and resolved by SDS-PAGE. Proteins were then analyzed by western blot using anti-p65 antibody. Blots were re-probed with anti-tubulin antibody as a loading control for cytosolic fractions and to confirm the purity of nuclear fractions. (B) Naïve (upper panels) and anergic (lower panels) T lymphocytes were stimulated for the indicated time points and NF-κB p65 localization was determined by immunofluorescence microscopy. Cells were co-stained with the nuclear dye SYTO-13. (C) Quantification of p65 nuclear localization shown in (B). Data show mean percentage of p65/SYTO-13 colocalization of 15 individual cells ± S.D., and are representative of three independent experiments.