Fig. 2. Interaction between p65 and 14-3-3 depends on phosphorylation. (a,b) Pull-down assay with GST-14-3-3 and cell lysates from TNF-treated HEK-293T cells incubated with acid phosphatase for 30 minutes (a) or from cells incubated for 2 hours with the indicated inhibitors plus 30 minutes with TNF (b). The presence of p65 in the precipitates was determined by western blot with anti-p65 antibody. Coomassie staining of GST proteins is shown in lower panel. Inputs represent 1/10 of the lysates. (c) In vitro kinase assay to test the capacity of total cell lysates, from untreated or TNF-treated HEK-293T cells, to phosphorylate GST-p65 peptides including the putative 14-3-3 binding domains. Upper panels show phosphorylated peptides by autoradiography. Coomassie staining of GST proteins is shown in lower panel. (d) Cell lysates from HEK-293T transfected with the full-length GFP-p65wt and the indicated mutant plasmids, treated for 60 minutes with TNF were precipitated with the anti-p65 antibody. Western blot analysis with anti-P-14-3-3 binding motif antibody confirmed the presence of phosphorylated 14-3-3 binding domains in p65. Precipitated GFP-p65 protein levels are shown in the lower panel. (e) Pull-down experiment using GST-14-3-3 and lysates from HEK-293T cells transfected with the indicated p65 mutants and treated for 30 minutes with TNF. Upper panel shows immunoblot with anti-p65. Coomassie staining of GST proteins is shown in lower panel. Inputs represent 1/10 of the lysates.