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. 2014 Jan 21;289(10):7082–7091. doi: 10.1074/jbc.M113.543769

FIGURE 3.

FIGURE 3.

NF-κBp50 mediates BAMBI repression by LPS and TNF-α. A and B, BAMBI mRNA levels in LX2 cells after 4 h of stimulation with LPS (100 ng/ml) (A) or TNF-α (10 ng/ml) (B) were determined by qPCR. Before treatment, LX2 cells were transfected with the Ad-GFP or Ad-IκB superrepressor overnight. Un, untreated. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01. C and D, BAMBI mRNA levels in hHSCs after 4 h of LPS (100 ng/ml) (C) or TNF-α (10 ng/ml) (D) treatment were determined by qPCR. Before treatment, hHSCs were transfected with control Ad-GFP or the Ad-IκB superrepressor overnight. Un, untreated. Data represent mean ± S.D. **, p < 0.01. E, after transfection with siRNA for p50 or a control for 72 h, LX2 cells were stimulated with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 4 h. The knockdown efficiency of siRNA p50 was evaluated by Western blot analysis for p50. BAMBI mRNA was measured by qPCR. Un, untreated. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01. F, LX2 cells were transfected with siRNA for p65 or a control for 72 h, followed by stimulation with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 4 h. p65 expression was evaluated by Western blot analysis. BAMBI mRNA was measured by qPCR. Un, untreated. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01. G, BAMBI luciferase reporter activity was measured. LX2 cells were cotransfected with −3384/+82-luc and siRNA p50. The Renilla luciferase reporter was used to normalize transfection efficiency. Data represent mean ± S.D. **, p < 0.01. H, two κB binding sites within −3384 to −1560 of the hBAMBI promoter region were identified using bioinformatics analysis. I, effects of LPS and TNF-α on the recruitment of p50 to the κB binding site in the hBAMBI promoter were assessed by ChIP analysis. LX2 cells (top panel) and hHSCs (bottom panel) were treated with LPS (100 ng/ml) or TNF-α for 2 h. After fixation, soluble chromatin was immunoprecipitated using anti-p50 or control Ab (normal mouse IgG). BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. J, effects of Ad-IκBsr on the recruitments of p50 to the κB binding site in the hBAMBI promoter were assessed. LX2 cells were treated with control Ad-GFP or Ad-IκBsr overnight and then treated with LPS (100 ng/ml) or TNF-α for 2 h. A ChIP analysis using anti-p50 or control Ab (normal mouse IgG) was performed. BAMBI promoter fragments containing the −3166 binding site were amplified by PCR. Un, untreated.