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. 2012 Oct 31;287(51):43019–43029. doi: 10.1074/jbc.M112.388694

FIGURE 2.

FIGURE 2.

p65 (21–186) truncated mutant localizes in the cytoplasm. A, whole cell lysates from HEK293T cells, transiently transfected with either EGFP or indicated EGFP fusion proteins, were immunoblotted directly with GFP antibody. B, immunofluorescence micrographs of HEK293T cells transfected as in A. The nuclei were counterstained with Hoechst 33342. The scale bar equals 10 μm. C, immunoblot analysis of cytosolic (Cyto) and nuclear (Nuc) subcellular fractions of HEK293T cells overexpressing EGFP or EGFP-tagged p65 (21–186). Hsp90 and poly(ADP-ribose) polymerase (PARP) serve as cytosolic and nuclear markers, respectively, and/or loading controls throughout. D, whole cell lysates (Input) from HEK293T cells, transfected with either full-length or 21–186 truncated mutant p65 fused to EGFP, were immunoblotted directly or after IP with GFP antibody for GFP fusion proteins and endogenous p65. The asterisk indicates nonspecific heavy chain (HC). E, immunoblot analysis of cytosolic (Cyto) and nuclear (Nuc) subcellular fractions of HEK293T cells overexpressing either EGFP-fused full-length or 21–186 truncated mutant p65 and stimulated with TNF (50 ng/ml) for the indicated periods. Caspase-3 and poly(ADP-ribose) polymerase serve as cytosolic and nuclear markers, respectively, and/or loading controls throughout. The results are representative of two to four experiments.