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

FIGURE 3.

FIGURE 3.

p65 (21–186) fragment selectively attenuates RPS3 nuclear translocation and gene transcription, without affecting p65 nuclear accumulation. A and B, HEK293T cells were transiently transfected with either EGFP vehicle control or EGFP-tagged p65 (21–186) truncated mutant, followed by stimulation with TNF (50 ng/ml) for the indicated period. Whole cell lysates were derived, separated, and immunoblotted directly for IκBα (A) or serine 32 and serine 36 phosphorylated IκBα (p-IκBα) (B) with β-actin serving as a loading control. C, immunoblot analysis of cytosolic (Cyto) and nuclear (Nuc) subcellular fractions of HEK293T cells that were transfected with either EGFP or EGFP-tagged p65 (21–186) mutant and stimulated with TNF (50 ng/ml) for indicated period. Hsp90 and poly(ADP-ribose) polymerase (PARP) served as cytosolic and nuclear markers, respectively, and/or loading controls. D, HEK293T cells, transfected with EGFP-tagged p65 (21–186) truncated mutant, were left untreated (Unstim) or stimulated with TNF for 30 min. Shown are micrographs of transfected EGFP-fused p65 (21–186), endogenous RPS3, and Hoechst 33342-counterstained nuclei. TNF-triggered RPS3 nuclear translocation was attenuated in the p65 (21–186) fragment transfected cells (indicated by filled triangles), compared with adjacent nontransfected cells. E, HEK293T cells were treated as in D. Shown are confocal micrographs of transfected EGFP-tagged p65 (21–186), endogenous p65, and Hoechst 33342-counterstained nuclei. TNF-triggered p65 nuclear translocation was identical in the p65 (21–186) truncated mutant transfected cells, compared with adjacent nontransfected cells (indicated by open triangles). F and G, Jurkat cells transiently transfected with either EGFP vehicle or EGFP-tagged p65 (21–186) truncated mutant were left untreated (Unstim) or stimulated with CD3 and CD28 antibodies (1 μg/ml each, αCD3/28) for 6 h (F) and 3 h (G), respectively. RT-PCR analysis of mRNA for IL2 (F) and CD25 (G) was normalized to expression of GAPDH and presented relative to the expression in untreated cells. *, p < 0.001 (F) and not significant (G) by Student's t test.