Figure 6

Analysis of concentration of multiple BMPs in bovine and human serum samples. (A and C) C2C12-BRE cells were treated as described in Figure 4 with three separate batches of FCS (bovine serum 1–3) or four different batches of human serum (human serum 1–4) with and without 5 ng/ml BMP4 antibody, 2 ng/ml BMP6 antibody and 0.1 ng/ml BMP9 antibody in separate wells. After 16 hours of treatment, cells were lysed and luciferase activity and protein content were determined and the relative luciferase units were calculated as described in Figure 2. Δ luciferase units for each BMP was calculated as described in Figure 4 and are displayed as part of the total luciferase activity obtained when serum was added in absence of blocking antibodies. Remaining luciferase activity induced by serum but not blocked by BMP4, BMP6 and BMP9 antibodies is labelled as BMPX. (B and D) By extrapolating the Δ luciferase units for each BMP in the appropriate standard curve, the amount of BMP4, BMP6 and BMP9 in each serum was calculated. Data shown is the mean ± SD of a representative experiment performed in quadruplicate.