BMP activity measured in biological samples. (A) 4 × 103 C2C12BRA cells/well were treated with supernatants of COS cells transfected with either a BMP-4 or a Dsl expression vector. Empty pcDNA3 plasmid was used as a mock control (B) 4 × 103 C2C12BRA cells/well were treated with supernatants of 293T cells transfected with the empty plasmid pcDNA3 (mock) or BMP-7 expression vector. (C) 4 × 103 C2C12BRA cells/well were cocultured with 4 × 103 293 cells/well stably transfected with a pcDNA3 (mock) or a BMP-7 expression plasmid. After 24 h, luciferase activity was measured. (D) 4 × 103 C2C12BRA cells/well were cocultured with 1 × 104 wt or Ltbp-4 hypomorphic (Ltbp-4-/-) lung fibroblasts. Luciferase activity was measured after 24 h. Conditioned medium from the coculture was collected from wt and Ltbp-4-/- lung fibroblasts and applied to C2C12BRA cells for 15 h to measure the amount of BMP activity. (E) 5 × 103 wt or Ltbp-4 hypomorphic (Ltbp-4-/-) lung fibroblasts were added to a 96 well plate. After 24 h, 4 × 103 C2C12BRA cells were added with or without 2 μg/ml of recombinant noggin. Luciferase activity was measured after 24 h. The results are expressed as relative luciferase units (RLU). Each point represents the mean ± SEM of triplicate wells from one representative experiment. Similar results were obtained in two separate experiments.