FIG. 3.

IκBαM overexpression inhibits p50/p65 NF-κB activation in NIH 3T3 and J774 cells. NIH 3T3 or J774 cells were transduced with either control (LXSN) or IκBαM retroviral vector. (A) To verify that the IκBαM-expressing cells were inhibited for p50/p65 NF-κB-mediated transcription, J774 and 3T3 cells were incubated with ±1 nM TNF or LTα for 24 h, respectively, and cell surface levels of ICAM-1 (a NF-κB responsive gene) were analyzed by flow cytometry. Dotted histograms represent binding of isotype control antibody, gray histograms are cells untreated with cytokine, and black histograms are cytokine-treated cells. (B) Translocation of p50/p65 NF-κB to the nucleus is inhibited in IκBαM-expressing cells. NIH 3T3 LXSN or IκBαM (+) cells were treated with LTα (1 nM) or MCMV (MOI = 2) for 2 h; nuclear extracts were prepared and subjected to EMSA. The arrow identifies the p50/p65 heterodimer of NF-κB. M, mock treated; cp, addition of unlabeled competitor, NF-κB-binding oligonucleotide probe.