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Fig. 6. 14-3-3 proteins associate with the chromatin in response to TNF ${alpha}$ to modulate NF ${kappa}$ B-dependent transcription. (a) Subcellular localization of endogenous 14-3-3 (left panels), p65 (middle panels) and I ${kappa}$ B ${alpha}$ (right panels) in control or HEK-293T cells treated for 30 minutes with TNF ${alpha}$ . PI was used for nuclear staining. Slides from three independent experiments were counted on the BX-60 microscope and the percentage of cells displaying nuclear 14-3-3 is indicated. Representative confocal images are shown (magnification, 630x). (b) Western blot with the indicated antibodies of total cell lysates (left) and nuclei (right) from HEK-293T cells incubated with TNF ${alpha}$ at different time points. Absence of ß-actin in the nuclear extracts is shown as a fractionation control whereas HDAC1 was detected as a nuclear loading control. (c) Chromatin from TNF ${alpha}$ -treated NIH-3T3 cells was immunoprecipitated with anti-14-3-3ß and - ${gamma}$ , anti-p65 and anti-I ${kappa}$ B ${alpha}$ antibodies. The presence of the indicated NF ${kappa}$ B target promoters in the precipitates was determined by PCR. (d) Chromatin from HEK-293T cells untransfected or transfected with DN14-3-3 and treated with TNF ${alpha}$ at different time points, was immunoprecipitated with the ${alpha}$ -p65 antibody. The presence of the indicated NF ${kappa}$ B target promoters in the precipitates was determined by PCR. (e) Semiquantitative RT-PCR to determine the transcriptional activity of the indicated NF ${kappa}$ B target genes in HEK-293T cells untransfected ( ${blacktriangleup}$ ) or transfected with DN-14-3-3 ( ${blacksquare}$ ) treated with TNF ${alpha}$ at the indicated time points. Graphs represent the relative amounts of mRNA, as measured by densitometric analysis, from one of two equivalent experiments.

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