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Fig. 4. 14-3-3 binding domains in p65 and I ${kappa}$ B ${alpha}$ are important to regulate their subcellular distribution. (a) GFP-p65 mutants were transfected in RPW1 cells to determine their subcellular localization. Slides from three independent experiments were counted on the BX-60 microscope and the percentage of cells displaying nuclear p65 is indicated. Representative confocal images are shown (magnification, 630x). (b) Immunolocalization of flag-p65 and the I ${kappa}$ B ${alpha}$ mutants in I ${kappa}$ B ${alpha}$ ^-/- MEF transfected with the indicated plasmids. Slides were counted on the BX-60 microscope and the percentage of cells displaying nuclear p65 and I ${kappa}$ B ${alpha}$ is indicated. Representative images are shown (magnification, 400x). (c) Cell lysates from HEK-293T cells cotransfected with HA-I ${kappa}$ B ${alpha}$ and the indicated GFP-p65 mutants were precipitated with anti-HA antibody. The presence of wild-type or mutant GFP-p65 in the precipitates was determined by immunoblotting with anti-p65 antibody. Input represents 1/10 of the lysates. (d) Anti-HA antibody was used to precipitate cell lysates from HEK-293T transfected with GFP-p65 and the indicated HA-I ${kappa}$ B ${alpha}$ mutants. Western blot ${alpha}$ -p65 is shown in the upper panel. Input represents 1/10 of the lysates.

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