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. 1999 Sep;155(3):841–851. doi: 10.1016/s0002-9440(10)65183-8

Figure 5.

Figure 5.

The effects of plasmin inhibitors on the expression of LAP, TGF-β1 and activation of L-TGF-β1. Immunoblot analysis of LAP and TGF-β1 antigens obtained from immunoprecipitates using anti-TSP-1 antibody (mAb133) and lysates of alveolar macrophages cultured in the absence or presence of α2-antiplasmin or aprotinin. A, top: Alveolar macrophages obtained 7 days after bleomycin administration cultured in the absence (lane 1) or presence of α2-antiplasmin at 10−4 units/ml (lane 2), α2-antiplasmin 10−1 unit/ml (lane 3), or aprotinin 0.01 μg/μm l (lane 4) were immunoblotted with anti-LAP antibodies and demonstrate protein bands compatible with L-TGF-β (100 kd) and the dimer (68 kd) of LAP. A, bottom: The same nitrocellulose filter was reprobed for immunoblotting with anti-TGF-β1 antibodies and demonstrates the dimer (25 kd) of TGF-β1. The culture conditions of alveolar macrophages used for lanes 1–4 are described above. The immunoblots are representative of experiments done 3 times. The numbers on the left denote the molecular weight of the bands in kilodaltons. B: Effects of aprotinin on the activation of L-TGF-β1 in the presence of sTSP-1. Alveolar macrophages obtained 7 days after bleomycin administration were cultured in the absence or presence of 0.01 μg/μm l/10 6 macrophages of aprotinin and/or in the absence or presence of 0.4 μg/ml/10 6 macrophages of sTSP-1. The TGF-β1 present in neutral and acidified then neutralized CM was quantitated and the percentage of active TGF-β1 was calculated. Each point is the mean of 3 to 7 rats. Percentage of active TGF-β1 in CM of alveolar macrophages from BLM-treated rats compared to normal saline N/S-treated rats or the CM of alveolar macrophages obtained after BLM treatment cultured with aprotinin have a P value ≤0.0001. Percentage of active TGF-β1 in CM of alveolar macrophages cultured in the presence of sTSP-1 compared to when no TSP-1 or aprotinin were present has a P value ≤0.0004. Percentage of active TGF-β1 in CM of alveolar macrophages obtained after BLM treatment and no in vitro treatment, compared to when sTSP-1 and aprotinin was also present, has a P value ≤0.0976. Percentage of active TGF-β1 in CM of alveolar macrophages cultured with sTSP-1 compared to when sTSP-1 and aprotinin were present has a P value <0.0001.