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. Author manuscript; available in PMC: 2010 Oct 28.
Published in final edited form as: J Neurosci. 2010 Apr 28;30(17):5843–5854. doi: 10.1523/JNEUROSCI.0137-10.2010

Figure 4.

Figure 4

Fibrinogen-bound latent TGF-β is activated by astrocytes. A, Fibrinogen-induced Smad2 phosphorylation is blocked by anti-TGF-β neutralizing antibody. B, Plasma-isolated fibrinogen was tested for active and latent TGF-β with TGF-β isoform-specific ELISAs. C, Delayed fibrinogen-induced Smad2 phosphorylation in primary astrocytes. Astrocytes were treated with fibrinogen or TGF-β (positive control) for the indicated times or left untreated, and lysates were blotted for P-Smad2. TGF-β induces Smad2 phosphorylation (P-Smad2) as early as 10 min, fibrinogen took 30 min. D, Western blotting of fibrinogen isolated from plasma reveals LAP1 and latent LTBP1. E, Plasma-isolated fibrinogen was immunoprecipitated with an antibody against human fibrinogen and immunoblotted with an antibody against LTBP1, revealing a complex of LTBP1 with fibrinogen. F, TGF-β ELISA revealed active TGF-β in supernatants of primary astrocytes treated with fibrinogen for 1 h. Results are from three independent experiments performed in duplicate. G, Immunolabeling for active TGF-β (green) and GFAP (red) revealed active TGF-β formation in astrocytes 1 h after fibrinogen treatment. H, Stereotactic injection of fibrinogen in mouse cortex results in active TGF-β formation, compared to control ACSF injection. Representative immunostaining is shown from (n=5 mice per group). Values are mean ± SEM. *p < 0.05. Scale bars: G,13.5 μm; H, 90 μm.