Molecules and Cells

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Fig. 4.

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Fig. 4. Smad3/HDAC2 complex inhibits NF-κB activation. (A) Co-immunoprecipitation (IP) using the Smad3 antibody. NCI-H292 cells were treated with the indicated concentrations of TGFβ1 for 30 min and subsequently lysed for IP assays. The immunoprecipitants collected by the Smad3 antibody were immunoblotted (IB) with an antibody against HDAC2 or Smad3. The input panels represent 4% of the cell extracts used for IP, immunoblotted using antibodies against phosphorylated HDAC2, Smad3, and phosphorylated Smad3. TGFβ-induced Smad3 phosphorylation increased in a concentration-dependent manner. Smad3 antibody was used as the loading control. The numbers at the bottom of each lane represent the relative band intensity normalized to the control (Smad3 without phosphorylation). (B) TGFβ1-induced colocalization of SMAD3 and HDAC2 proteins in the cell nucleus (closed and open arrowheads, respectively). NCI-H292 cells were co-labeled using antibodies against Smad3 (green) and HDAC2 (red). The cell nucleus was stained with DAPI (blue). The merged confocal images were shown at the bottom. SIS3 (1 µM), a specific Smad3 inhibitor, was treated to suppress the nuclear colocalization of Smad3 or HDAC2 (asterisks). (C) The acetylated level at lysine 310 of NF-κB (Ac-NF-κB (K310)) was reduced by TGFβ1 (arrows) and restored by an additional Smad3 inhibitor, SIS3 (asterisk). NCI-H292 cells were stained with anti-Ac-NF-κB (K310) antibody (green). Cell nuclei were stained with DAPI (blue). Lower histogram: Quantification of the confocal images. The signal intensity for Ac-NF-κB (K310) in the cell nucleus was normalized to the control (without TGFβ1 treatment). The bar graph represents the mean ± SD of three independent experiments (**P < 0.01). (D) ChIP assay using anti-HDAC2, anti-NF-κB, or anti-Ac-NF-κB antibody on the MUC5AC promoter region. NCI-H292 cells were treated with each indicated concentration of TGFβ1 for 30 min. PCR primers were designed to identify the Smad3 RE (–234 to –134 bp) or NF-κB RE (–349 to –145 bp) within the MUC5AC promoter region. Upper panels: The recruitment of HDAC2 to the Smad3 RE on the MUC5AC promoter was increased by TGFβ1 addition in NCI-H292 cells. Middle panels: The binding of NF-κB to the MUC5AC promoter seemed not to be affected by TGFβ1 addition. Lower panels: Contrary to NF-κB binding to the MUC5AC promoter, epigenetic modification on NF-κB (Ac-NF-κB at K310) was prominently reduced by TGFβ1. Rabbit IgG was used as a negative control. Input samples (unprecipitated chromatin) were used as positive controls for PCR amplification.
Mol. Cells 2021;44:38~49
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