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. 2005 Oct 10;391(Pt 2):399–408. doi: 10.1042/BJ20050795

Figure 10. Effect of TNFα on c-Jun and Nrf2 binding to the GCLM ARE (A, B) and importance of the ARE in TNFα-mediated induction of GCLM promoter activity (C).

Figure 10

H4IIE cells were treated with TNFα (15 ng/ml, 4 h) or vehicle control and subjected to EMSA with supershift (A) or ChIP analysis (B) as described in the Materials and methods section. (A) EMSA with supershift results: note that TNFα treatment increased the nuclear binding activity of both Nrf2 and c-Jun to the ARE of the rat GCLM. Arrows in (A) point to supershifted bands. 100X Comp, 100-fold unlabelled probe. (B) H4IIE cells were treated with TNFα or vehicle control, then processed for ChIP assay as described in the Materials and methods section. PCR products from amplification of the ARE site after immunoprecipitation with antisera against Nrf2 or c-Jun demonstrate that TNFα treatment led to increased Nrf2 and c-Jun binding to the ARE site. Input genomic DNA (gDNA input) was used as a positive control and a no antibody immunoprecipitation (no Ab) was used as a negative control. Representative results from two experiments are shown. (C) The functional significance of the ARE element. To confirm functionality of the ARE, EMSA was first performed to document the effect of site-directed mutagenesis on nuclear-binding activity (C, left panel, the number of mutated bases is shown on top; note that nuclear binding was completely abolished only when three bases were mutated). The right panel shows the effect of preventing ARE binding on baseline and TNFα-mediated increase in GCLM promoter activity. H4IIE cells were transfected with wild-type (WT) or mutated (MUT, mutated in three bases) GCLM −329/+3-LUC constructs and treated with TNFα (15 ng/ml, 4 h). Results represent means±S.E.M. from five to nine experiments (C, right panel). Data are expressed as relative luciferase activity compared with that of pGL-3 basic vector control, which is assigned a value of 1.0. Note that the mutant construct had lower baseline promoter activity and TNFα no longer was able to induce its reporter activity. *P<0.001 versus WT −329/+3-LUC (ANOVA followed by Fisher's test).