FIG 6.

Interaction of p65 with IκBα or chromatin in the nucleus. (A) HeLa cells cultured in 6-cm dishes were transfected with 2 μg pCHA, HA-SET-Nup214, or HA-DEK-Nup214. At 2 days after transfection, cells were collected and were subjected to IF assays and PLAs. Anti-p65 (ab7970; dilution, 1:100; Abcam) and anti-IκBα (L35A5; dilution, 1:30; Cell Signaling Technology, Inc.) antibodies were used as primary antibodies. “Merged” panels show composite images of cells stained with Alexa Fluor 488, Detection Reagents Red (for PLA), and Alexa Fluor 633. Bars, 10 μm. (B) HEK293T cells were transfected with pCAGGS, pCAGGS-SET-Nup214 (SN214), or pCAGGS-DEK-Nup214 (DN214) (0.2 or 2 μg) and were incubated for 2 days. Cells were collected, and IP assays were conducted using anti-p65 (ab7970) and rabbit polyclonal IgG antibodies (PP64B) (Merck KGaA, Germany). Proteins in input lysates and immunoprecipitated samples were separated by 10% or 5% SDS-PAGE, and Western blot analyses were performed using anti-p65, anti-IκBα, anti-Nup214, and anti-C23 antibodies. Molecular weights (in thousands) of prestained markers are indicated on the right. (C) HEK293T cells were transfected with 5 μg pCAGGS, pCAGGS-SET-Nup214 (SET-N214), or pCAGGS-DEK-Nup214 (DEK-N214). At 2 days after transfection, cells were either left untreated (−) or treated with TNF-α (20 ng/ml) for 30 min; they were then subjected to ChIP assays using 2 μg anti-IgG or anti-p65 (ab7970) antibodies to measure the binding of p65 to A20 or IκBα promoter regions. The levels of immunoprecipitated DNA were then normalized to the input DNA level. Results are shown as fold activation relative to the level of DNA immunoprecipitated from pCAGGS-transfected lysates by the anti-p65 antibody in the absence of TNF-α. Data are means ± standard deviations for three independent experiments. *, P < 0.05.