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. 2014 Apr 23;289(22):15244–15258. doi: 10.1074/jbc.M113.540633

FIGURE 5.

FIGURE 5.

IκBα mediates the activation of NRF2 and NF-κB induced by RAC1. A, a dominant-negative mutant of IκBα (IκBαS32A/S36A) inhibits RAC1-dependent induction of the NF-κB pathway. HEK293T cells were co-transfected with HIV-LUC reporter, RAC1Q61L, IκBαS32A/S36A, or both in the absence (white bars) or presence (black bars) of p65/p50. B, IκBα is implicated in the induction of NRF2 by RAC1. Cells were co-transfected with ARE-LUC promoter, RAC1Q61L, IκBαS32A/S36A, or both in the absence (white bars) or presence (black bars) of p65/p50. One-way ANOVA followed by a Newman-Keuls post-test was used to assess significant differences among groups. Asterisks denote statistically significant differences with: *, p < 0.05; **, p < 0.005; ***, p < 0.001; ###, p < 0.001 (with respect to their control). C, Western blot analysis of cells co-transfected with RAC1Q61L, IκBαS32A/S36A, and NRF2ΔETGE-V5 and their respective controls. Upper panel, immunoblot with anti-V5 antibody; middle panel, immunoblot with anti-AU5; lower panel, anti-GAPDH as protein load control. D, EMSA, using a double-stranded oligonucleotide containing the core ARE sequence and nuclear extracts from HEK-TLR4-MD2/CD14 cells treated with 1 μg/ml LPS for 1 h. E, EMSA, using a double-stranded oligonucleotide containing the core ARE sequence and nuclear extracts from HEK293T cells expressing RAC1Q61L ± IκBαS32A/S36A. F, EMSA, using a double-stranded oligonucleotide containing the core ARE sequence and nuclear extracts from HEK293T cells expressing RAC1Q61L ± DN-NRF2. In all cases the concentration of unlabeled oligonucleotide was 100× in the competition binding between labeled and unlabeled ARE probe. G, EMSA, using a double-stranded oligonucleotide containing the NF-κB binding element and nuclear extracts from HEK-TLR4-MD2/CD14 cells treated with 1 μg/ml LPS for 1 h. H, EMSA, using a double-stranded oligonucleotide containing the NF-κB promoter sequence and nuclear extracts from HEK293T cells expressing RAC1Q61L ± IκBS32A/S36A. I, EMSA, using a double-stranded oligonucleotide containing the NF-κB promoter sequence and nuclear extracts from HEK293T cells expressing RAC1Q61L ± DN-NRF2. In all cases the concentration of unlabeled oligonucleotide was 100× in the competition binding between labeled and unlabeled NF-κB probe. IkB* stands for IkB S32A/S36A mutations (a mutated plasmid). + indicates that the complete sample is Rac1Q61L + DN-NRF2, for example.