Effect of I3C on the TNF-induced translocation of p65 into the nucleus. (A) Western blot analysis of phospho-p65 and p65 using cytoplasmic extracts (CE). Jurkat cells were incubated with 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times in minutes. Cytoplasmic extracts were prepared and subjected to Western blot analysis using anti-p65 antibody and phosphospecific anti-p65 antibody. (B) Western blot analysis of phospho-p65 and p65 using nuclear extracts (NE). Jurkat cells were incubated with 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Nuclear extracts were prepared and subjected to Western blot analysis using anti-p65 and phosphospecific anti-p65 antibodies. For loading control of nuclear protein, the membrane was blotted with anti-PARP antibody. (C) Effect of I3C on TNF-induced acetylation of p65. Jurkat cells were incubated with 50 μM I3C for 24 hours and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared, immunoprecipitated (IP) with anti-p65 antibody, and then examined by Western blot analysis (WB) using anti-acetyl-lysine antibody. Whole-cell extracts were subjected to Western blot analysis using anti-p65 antibody. (D) Immunocytochemical analysis of p65 localization after treatment with 1 nM TNF in the absence or presence of 50 μM I3C. Jurkat cells were incubated with I3C for 24 hours and then treated with 1 nM TNF for 30 minutes. Cells were subjected to immunocytochemistry as described in “Materials and methods.” Stained slides were mounted with mounting medium (Sigma-Aldrich) and analyzed under an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and MetaMorph Version 4.6.5 software (Universal Imaging, Downingtown, PA). Original magnification, ×200.