Anti bmp6

For neutralization of BMPs, cells were treated with 1 or 3 μg/mL anti-BMP4, anti-BMP6, anti-BMP7, (R&D Systems, Minneapolis, MN, USA cat#MAB757, MAB507, MAB3541) or 3 μg/mL isotope-matched anti-IgG (R&D systems, cat#MAB004) for 48 hours. For neutralization of hepcidin, spheroids were treated during time of plating with 1 or 3μg/mL of anti-Hepcidin-25 (Amgen, Thousand Oaks, CA (19D12)(71 (link)); referred to as αHep#1), anti-Hepcidin-25 (Abcam, Cambridge, MA, USA cat# ab30760; referred to as αHep#2); or anti-IgG (R&D systems, cat#MAB004) for 48 hours. For neutralization of IL-6 in TAF conditioned media, TAF conditioned media was collected as described above and pre-treated with 1 or 5 μg/mL anti-IL-6 or isotope-matched anti-IgG neutralizing antibodies (R&D Systems, cat#MAB2061-100, MAB004) for one hour before addition of CM to TECs during spheroid plating. Human recombinant IL-6 (R&D Systems) was used at 2 and 200 ng/mL. Human recombinant GDF-15 (R&D Systems) was used a 0.1, 0.5, 1, 5, 10, 100 and 200 ng/mL.

Cells were treated with 1 or 3 μg/ml anti-BMP4, anti-BMP6, anti-BMP7, or anti-IL6 neutralizing antibodies (R&D Systems). 3 μg/ml Isotope matched anti-IgG was used as a control (R&D Systems). Human recombinant BMP6 (R&D systems), BMP4 and BMP7 (GIBCO) were used at 50 – 200 ng/ml for 24 hrs. Cells were treated with human recombinant IL6 (Peprotech) for 24 hours.