Figure 7.

TNF-α-induced activation of CHGA Hap2 promoter via c-Rel. A, IMR-32 cells were transfected with CHGA promoter Hap2–GLuc and Hap5–GLuc constructs and treated with increasing doses of TNF-α for 6 h. Results are mean ± S.E. of triplicate values of Gaussia luciferase/μg of protein. **, p < 0.01 and ****, p < 0.0001 when compared with basal condition and #, p < 0.05 and ####, p < 0.0001 when compared with corresponding treatments with the Hap5 construct. B, effect of TNF-α treatment on c-Rel expression. IMR-32 cells were treated with increasing doses of TNF-α for 6 h and Western blot analysis of total proteins was carried out probing for c-Rel and GAPDH. C and D, role of c-Rel in haplotype-specific activation of CHGA promoter Hap2 by TNF-α. CHGA promoter Hap2–GLuc and Hap5–GLuc constructs were transfected into IMR-32 cells with or without c-Rel expression plasmid (C) or c-Rel siRNA oligos (D) followed by treatment with TNF-α (5 ng/ml) for 6 h. Results are mean ± S.E. of triplicate values of Gaussia luciferase/μg of protein. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001 when compared with the corresponding basal condition; ##, p < 0.01 and ####, p < 0.0001 when compared with the corresponding Hap5 construct conditions. Down-regulation of c-Rel was confirmed by Western blot analysis (shown in the bottom panels). E and F, ChIP assay of N2a cells transfected with CHGA promoter Hap2 or Hap5 constructs, with or without TNF-α treatment was carried out using antibody against c-Rel/IgG. Immunoprecipitated chromatin was subjected to qPCR. Fold-enrichment in signal obtained in the case of the c-Rel antibody over preimmune IgG is shown. **, p < 0.01 and ****, p < 0.0001 when compared with fold-enrichment in untreated condition and ##, p < 0.01 and ####, p < 0.0001 when compared with the corresponding treatment in case of Hap2 construct.