FIG. 7.

NF-κB activity in response to DoxR. (A) IKK1/2^−/− MEFs were treated with indicated doses of DoxR for 12 h and analyzed for gel shift activity by using a NF-κB consensus oligonucleotide (left panel) or Oct-1 oligonucleotide (middle panel). Supershift analysis with antibodies to p50, p65, and p52 was performed on extracts derived from cells treated with 0.7 μg of DoxR/ml (right panel). (B) Construction of LV-κBluc and LV-Mut-κBluc vectors. The positions of the κB binding sites, along with the TATA box, have been indicated. (C) IKK1/2^−/− MEFs were transduced with LV-κBLuc, and pools of these cells were treated for 18 h with DoxR (0.3 μg/ml), TNF-α (10 ng/ml), and LPS (30 ng/ml). Luciferase activity was evaluated in a 96-well plate reader. (D) IKK1/2^−/− MEFs were either transduced with LV-κBLuc or LV-Mut-κBluc vectors, and pools of these cells were treated for 18 h with DoxR before luciferase activity was measured. (E) Real-time PCR analysis for IκBα. IKK1/2^−/− MEFs were treated with the indicated doses of DoxR, and the levels of IκBα and actin messages were determined by real-time PCR analysis. The ratios of IκBα levels to actin levels were quantitated and are indicated as a fold change on the y axis.