Recombinant bmp9

Cells were stimulated in 0% FCS with recombinant BMP9 (R&D Systems) at the concentration indicated for 1 h after 1 h30 of serum deprivation. Cell extracts were lysed in 50 mmol/L Tris-HCl pH 7.4, 0.5 mol/L NaCl and a cocktail of protease inhibitors (Sigma) by sonication. 20 μg of proteins from cell lysates were separated on a SDS/PAGE, 4–20% (Bio-Rad) and analyzed by immunoblotting with anti-pSmad1/5/9 antibody (Cell Signaling #9511). The same membrane was reprobed with a monoclonal antibody against β-actin (clone AC-15; Sigma) to confirm equal protein loading.

The mouse fetal lung mesenchymal MFLM-91U cell line was obtained from Seven Hill Bioreagents. MFLM-91U cells were transfected with siRNAs or treated with recombinant BMP9 (25 ng/ml, R&D) and harvested 48 h after the treatment. After removal of culture medium by aspiration, the cells were washed with PBS and then fixed in 4% paraformaldehyde (PFA) for 15 mins. Cells were permeabilized with 0.1% Triton-X100 and incubated in blocking buffer (10% fetal bovine serum in PBS). The cells were stained with primary antibodies overnight at 4 °C, followed by 1-h incubation with Alexa-488 or Alexa-594-conjugated donkey anti-rabbit IgG (1:1000, Invitrogen, A-21207) and donkey anti-rat IgG (1:1000, Invitrogen A-21208). Cell nuclei were counterstained with Hoechst 33258 (Thermofisher). Images were acquired using Nikon Eclipse Ti2 inverted microscope. Lung cryosections were either stained with Hematoxylin and Eosin (H&E) or used for immunostaining as described^{55 (link)–58 (link)}. Antibodies for immunostaining are listed in Supplementary Table 7 and the staining conditions are described in^{59 (link)–62 (link)}. Morphometrical measurements of alveolar sizes and intra-vascular labeling with isolectin B4 were described in published studies^{14 (link),63 (link),64} . Co-localization studies and confocal imaging of lung tissue sections were performed as described^{65 (link)–67 (link)}.

ELISAs were used to measure IgG2a, IgG2b, and anti-BMP9 Ab levels in mouse serum. IgG2a and IgG2b ELISAs were performed as per the manufacturer’s instructions (eBioscience). For the anti-BMP9 Ab ELISA, 96-well ELISA plates (Maxisorp, Nunc) were coated with 100 μL of 1 μg/mL recombinant BMP9 (R&D Systems) in coating buffer (15 mM K₂HPO₄, 25 mM KH₂PO₄, 0.1 M NaCl₂, 0.1 mM EDTA, 7.5 mM NaN₃) and incubated overnight at 4 °C. Plates were then washed 3 times with 0.05% Tween PBS (PBST) and blocked for 1 h at room temperature (RT) with 1% BSA in PBS. After washing 3 times with PBST, serial dilutions of individual mouse serum samples and reference mouse anti-BMP9 Ab (MAB3209, R&D Systems) (diluted in 1% BSA PBS) were prepared and 100 μL/well were incubated for 2 h at RT. After 3 more washes, 100 μL/well horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobins (Igs) secondary Ab (Southern Biotech, diluted 1:500 in 1% BSA PBS) was incubated for 1 h at RT. TMB substrate was added after 5 washes and the reaction was allowed to develop for 30 min at RT. The optical density was measured at 450 nm using a TECAN GENios Pro plate reader.