Fig 1: Mouse eosinophil-associated-ribonuclease-1 uses bone morphogenetic protein receptors to activate the smad-1/5/8-Runt-related transcription factor-2 signalling pathway. (A) Wild-type and Runt-related transcription factor-2 KO mouse smooth muscle cell were exposed to osteogenic media with or without mouse eosinophil-associated-ribonuclease-1, interleukin-4, interleukin-13, eosinophil lysates from wild-type, Il4-/- or Il13-/- mice. Immunoblots of different bone morphogenetic protein receptors, TGFßRs, and phosphorylated Smad-2, Smad-3, and Smad-1/5/8. (B–G) Wild-type smooth muscle cell were exposed to osteogenic media for 14 days and stimulated with mouse eosinophil-associated-ribonuclease-1 for 30 min before collection. Immunoprecipitation with anti-mouse eosinophil-associated-ribonuclease-1 (B), anti-bone morphogenetic protein receptor-1A (C, top) and anti-bone morphogenetic protein receptor-1B (C, bottom) antibodies, followed by immunoblotting detection of different bone morphogenetic protein receptors, TGFßRs, or mouse eosinophil-associated-ribonuclease-1. (D) FITC-mouse eosinophil-associated-ribonuclease-1 (0~10.0 nM) binding affinity and Scatchard plot on smooth muscle cell treated with or without bone morphogenetic protein receptor siRNA or excessive BMP2 (1000 ng/mL). Immunofluorescence double-staining of mouse eosinophil-associated-ribonuclease-1 with bone morphogenetic protein receptor-1A (E), bone morphogenetic protein receptor-1B (F) or bone morphogenetic protein receptor-2 (G).
Fig 2: Binding of eosinophil cationic protein and eosinophil-derived neurotoxin on human smooth muscle cell bone morphogenetic protein receptors. Human smooth muscle cell were exposed to osteogenic media for 14 days and stimulated with recombinant eosinophil cationic protein or eosinophil-derived neurotoxin for 30 min before collection. Immunoprecipitation with anti-eosinophil cationic protein (A), anti-bone morphogenetic protein receptor-1A (B) and anti-bone morphogenetic protein receptor-1B (C) antibodies, followed by immunoblotting detection of different bone morphogenetic protein receptors, TGFßRs, and eosinophil cationic protein. (D) FITC-eosinophil cationic protein (0~10.0 nM) binding affinity and Scatchard plot on smooth muscle cell treated with or without bone morphogenetic protein receptor siRNA or excessive BMP2 (1000 ng/mL). Immunofluorescence double-staining of eosinophil cationic protein and bone morphogenetic protein receptor-1A (E) or bone morphogenetic protein receptor-1B (F). Immunoprecipitation with anti-eosinophil-derived neurotoxin (G), anti-bone morphogenetic protein receptor-1A (H), and anti-bone morphogenetic protein receptor-1B (I) antibodies, followed by immunoblotting detection of different bone morphogenetic protein receptors, TGFBRs, and eosinophil-derived neurotoxin. (J) FITC-eosinophil-derived neurotoxin (0~10.0 nM) binding affinity and Scatchard plot on smooth muscle cell treated with or without with bone morphogenetic protein receptor siRNA or excessive BMP2 (1000 ng/mL). Immunofluorescence double-staining of eosinophil-derived neurotoxin and bone morphogenetic protein receptor-1A (K) or bone morphogenetic protein receptor-1B (L).
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