Figure 3.
Nuak2 is a direct TGFβ target gene. A, cloning of the mouse Nuak2 promoter and enhancer. Shown is a schematic diagram of the mouse Nuak2 gene spanning the promoter sequences, the transcriptional start site (TSS), the first exon, and part of the first intronic sequence. B, ChIP assays using an antibody against endogenous SMAD2/SMAD3 (S2/3) and amplification of genomic sequences corresponding to the Nuak2 intronic enhancer, the Pai1 promoter, and the β-globin (Hbb) control region in NMuMG cells stimulated with or without 5 ng/ml TGFβ for 1 h. Control immunoprecipitations with mouse IgG are also shown as reference. The amount of PCR-amplified DNA signal after ChIP is normalized against the equivalent PCR signal of the input chromatin prior to immunoprecipitation, and the relative ratios are shown in the diagrams as average values determined from triplicate determinations with their corresponding S.E. (error bars). C, the genomic fragments, depicted in A, that were cloned into the luciferase reporter are shown relative to the luciferase (luc) cDNA in the corresponding constructs. D, luciferase assay from HEK 293T cells transiently transfected with the Nuak2 enhancer construct and pcDNA3 control or constitutively active pcDNA3-ALK5TD. E, luciferase assay was performed using NMuMG cells transiently transfected with the Nuak2 1- or 2-kbp promoters and the Nuak2 intronic enhancer constructs with or without TGFβ (5 ng/ml) stimulation for 17 h. Each independent experiment was repeated at least three times. Asterisks depict differences compared with respective controls or between the conditions indicated with lines: *, p < 0.05; **, p < 0.01; ***, p < 0.001.