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Fig 3. I ${kappa}$ B ${alpha}$ binds to 14-3-3 proteins independently of TNF ${alpha}$ . (a) Pull-down experiment using GST-14-3-3 ${eta}$ and lysates from HEK-293T cells incubated with TNF ${alpha}$ as indicated. Upper panel shows immunoblot with ${alpha}$ -p65 and ${alpha}$ -I ${kappa}$ B ${alpha}$ antibodies. Ponceau staining of GST proteins is shown in lower panel. Inputs represent 1/10 of the lysates. (b) Immunoprecipitation with anti-HA from control HEK-293T cells and cells transfected with HA-I ${kappa}$ B ${alpha}$ _32-36, treated for 60 minutes with TNF ${alpha}$ . The presence of co-precipitated endogenous 14-3-3 and p65 was determined by western blot with anti-14-3-3 ${gamma}$ / ${eta}$ or anti-p65 antibodies. (c) Pull-down assay with GST-14-3-3 ${eta}$ and cell lysates from HEK-293T cells untreated or treated with acid phosphatase for 30 minutes. The presence of I ${kappa}$ B ${alpha}$ proteins in the precipitates was determined by immunoblotting with anti-I ${kappa}$ B ${alpha}$ antibody. Ponceau staining of GST proteins is shown in the lower panel. Inputs represent 1/10 of the lysates. (d) Pull-down experiment using different GST-I ${kappa}$ B ${alpha}$ constructs and lysates from HEK-293T cells transfected with myc-14-3-3 ${eta}$ . Upper panel shows immunoblot with anti-myc antibody. Coomassie staining of GST proteins is shown in middle panel. (e,f) Pull down experiments using GST-14-3-3 ${eta}$ and lysates from HEK-293T cells transfected with the indicated Ha-I ${kappa}$ B ${alpha}$ deletion mutants. Upper panels show immunoblot with ${alpha}$ -HA. Coomassie staining of GST proteins is shown in the lower right panels. Inputs represent 1/10 of the lysates. A schematic overview of the I ${kappa}$ B ${alpha}$ protein is shown in d-f, indicating the putative 14-3-3 binding domain.

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