RelA - PubMed (original) (raw)

Down-modulation of Smad7 releases TGF-β/SMAD signaling from suppression by TNF-α. (A) Indirect immunofluorescence labeling with anti-Smad7 antibody (a,b,c,d) and anti-Smad2 antibody (e,f) in RelA+/+ fibroblasts cotransfected with constructs expressing Smad7 antisense mRNA (pSmad7AS) and pEGFP. (a,b,e) Red fluorescence signal for Cy3-conjugated secondary anti-rabbit (a,b) and anti-mouse IgG (e) only; (c,d,f) identical fields with red and green (GFP) fluorescence merged to identify transfected cells. (Arrows) Staining for endogenous Smad7 (a,b) and Smad2 (c) in untransfected cells. (Arrowheads) Transfected cells that are identified by green fluorescence (EGFP) and reveal decreased expression of Smad7 (a,b), but normal expression of Smad2 (e), respectively. (B) EGFP fluorescence (a,b), indirect immunofluorescence staining for anti-Flag (c,d), and merged fluorescence image (e,f) in RelA+/+ fibroblasts cotransfected either with Flag-tagged Smad3 (pFSmad3) and empty vector (pcDNA3) (a,c,e), or with Flag-tagged Smad3 (pFSmad3) and Smad7 antisense mRNA expression vector (pSmad7AS) (b,d,f). (C) Anti-Flag immunoblotting of Flag Smad7 in COS cells cotransfected with Flag-Smad7 plasmid (pF-Smad7) and empty vector DNA (pcDNA3) (lane 1), or with increasing amounts of Smad7 antisense plasmid (pSmad7AS) (lanes 2,3). (D) RelA+/+ fibroblasts were cotransfected with either 3TPLux reporter vector (histogram a), or SBE4 Luc reporter vector (histogram b), together with either empty control vector pcDNA3, or the same vector expressing Smad7 antisense mRNA (pSmad7AS) as indicated. Cells were incubated with or without TNF-α for 45 min prior to treatment with or without TGF-β1 for an additional 4 hr. Results are expressed as ratios (Fold-induction) of normalized luciferase activities in treated and untreated cells. Bars, mean ±

s.d.

of three independent experiments, respectively.