total smad protein  (Upstate Biotechnology Inc)

Journal:

Article Title: Imatinib mesylate inhibits the profibrogenic activity of TGF-? and prevents bleomycin-mediated lung fibrosis

doi: 10.1172/JCI200419603

Figure Lengend Snippet: TGF-β2 activation of c-Abl signaling and TGF-β2 activation of Smad signaling are independent. (A) Left: Smad3 KO MEFs (Smad3–/–) were transfected with Flag-tagged c-Abl (c-Abl–Flag) and dominant negative Smad2 (myc-Smad2D450E) as described in Methods. NIH-3T3 cells were treated as described in Figure ​Figure1.1. Following 30-minute stimulation in the absence (–) or presence (+) of 10 ng/ml TGF-β2, kinase activity of the transfected (c-Abl–Flag activity) or endogenous (c-Abl activity) c-Abl protein was determined using anti-Flag or K12 serum respectively. The corresponding total protein is indicated in the second and fourth panels. Right: NIH-3T3 or Smad2/3 KO cells were treated with (+) or without (–) TGF-β as described for the left panels. Following 30-minute stimulation, lysates containing equivalent protein were Western-blotted for the indicated proteins. (B) Left: NIH-3T3 cells were treated with (+) or without (–) TGF-β2 and/or imatinib as described in Figure ​Figure1.1. Following 30-minute stimulation, lysates containing equivalent protein were Western-blotted for phospho-Smad2 (p-Smad2), phospho-Smad3 (p-Smad3), or the corresponding total protein. Right: Abl–/–Arg–/– MEFs or Abl–/–Arg–/– fibroblasts stably expressing (+) WT-c-Abl or dominant negative c-Abl were treated with (+) or without (–) TGF-β2 for 30 minutes and processed for the indicated Smad protein as described for the left panels.

Article Snippet: The same blot was stripped and probed for total Smad protein (Smad2, no. 06-829; Upstate Biotechnology Inc.; and Smad3, no. 51-1500; Zymed Laboratories Inc.).

Techniques: Activation Assay, Transfection, Dominant Negative Mutation, Activity Assay, Western Blot, Stable Transfection, Expressing

Journal:

Article Title: Imatinib mesylate inhibits the profibrogenic activity of TGF-? and prevents bleomycin-mediated lung fibrosis

doi: 10.1172/JCI200419603

Figure Lengend Snippet: TGF-β stimulates c-Abl–dependent signaling. (A) IMR90 cells were treated with DMEM alone (–) or containing 10 ng/ml TGF-β2 (+). Following 30-minute stimulation, c-Abl kinase activity or Smad2 (p-Smad2) and Smad3 (p-Smad3) phosphorylation was determined. Total c-Abl, Smad2, and Smad3 protein is shown below the corresponding activity. (B) Top blots: IMR90 cells, Abl–/–Arg–/– MEFs, Abl–/–Arg–/– fibroblasts stably expressing (+) WT-c-Abl or dominant negative c-Abl, or PDGF-α and -β receptor–null F cells (PDGFR–/–) were left untreated (–) or treated (+) for 24 hours with TGF-β2 (10 ng/ml) and/or imatinib (10 μg/ml). Imatinib was added 20 minutes before TGF-β2, and fibronectin mRNA accumulation was determined. Bottom blots: Ethidium bromide staining for 28S and 18S ribosomal RNA subunits prior to Northern analysis. Top left graph: The fold fibronectin mRNA stimulation by TGF-β2 is indicated for the various cell types. Results represent the mean ± SE of 2 separate experiments. Bottom left graph: Cell lines as described above were transfected with a fibronectin luciferase construct. The fold induction of 10 ng/ml TGF-β2 with or without 5 μg/ml imatinib was determined as described in Methods and by Penheiter et al. (51). Right graphs: Collagen I (top) and collagen III (bottom) luciferase activity was determined as described for the bottom left graph. Results for all luciferase assays represent the mean ± SE of 2 separate experiments, each done in triplicate.

Article Snippet: The same blot was stripped and probed for total Smad protein (Smad2, no. 06-829; Upstate Biotechnology Inc.; and Smad3, no. 51-1500; Zymed Laboratories Inc.).

Techniques: Activity Assay, Stable Transfection, Expressing, Dominant Negative Mutation, Staining, Northern Blot, Transfection, Luciferase, Construct