Fig. 5. 14-3-3 activity is required to maintain p65 in the cytoplasm. (a) Immunolocalization of endogenous p65 in HEK-293T cells transfected with the indicated 14-3-3 plasmids and incubated with TNF for 15 and 60 minutes as indicated. Representative confocal images are shown (magnification, 630x). Western blot showing the levels of transfected wild type (WT) and DN-14-3-3 (DN) with anti-myc antibody. (b) Pull-down experiment using GST-14-3-3 and lysates from HEK-293T cells untransfected or transfected with DN-14-3-3 treated for 30 minutes with TNF as indicated. Upper panels show immunoblot with anti-p65 antibody. Inputs represent 1/10 of the lysates. Ponceau staining of GST proteins is shown in lower panel. (c) Nuclear extracts from HEK-293T cells, untransfected or transfected with DN-14-3-3 and treated with TNF at different time points, were precipitated with the anti-p65 antibody. Co-precipitated IB was detected by western blot with -IB antibody. Tubulin was detected as a fractionation control and HDAC1 as a loading control for nuclear extracts in the left panel. Inputs represent 1/10 of the lysates. (d) Pull-down experiment using GST-14-3-3 and lysates from wild-type or IB-knockout MEF cells untreated or treated for 30 minutes with TNF as indicated. Upper panel show immunoblot with anti-p65 antibody. Ponceau staining of GST proteins is shown in middle panel. Inputs represent 1/10 of the lysates (detected with anti-p65). (e) Subcellular localization of endogenous p65 in HEK-293T cells treated with siRNA against different 14-3-3 isoforms. Right panels show the nuclear entry of HDAC4 in the 14-3-3- siRNA-treated cells as a control. Representative confocal images are shown (magnification, 630x). PI was used for nuclear staining. Western blot assayed with anti-pan-14-3-3 antibody recognizing 14-3-3 family members shows the levels of 14-3-3 in cell lysates from HEK-293T cells treated with the isoform-specific siRNA. HDAC1 was detected as a loading control.