Alk1 fc

HUVECs were isolated from anonymous umbilical veins, as described before72 (link). HUVECs were subcultured using a trypsin/EDTA-reagent pack (Lonza) and maintained in 5% fetal bovine serum (FBS)-containing EC growth medium (Sciencell). Cells were treated or not for 24 h with 1 μg/mL ALK1-Fc (R&D Systems) in complete EC growth medium containing 5% FBS. Cells were then rinsed with PBS and processed for RNA extraction and RNA-Seq as described above. For WB analyses, cells were processed as before73 (link) with the following modifications. Cells were solubilized in RIPA buffer (EMD Millipore) supplemented with 1× Complete protease inhibitor mixture (Roche Applied Science). 5–20 μg of proteins (depending on the primary Ab used) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were then probed with Abs directed against phospho-Smad1/5/8 (Cell Signaling Technology), total Smad5 (Cell Signaling Technology), ID1 (BioCheck), ANG2 (Santa Cruz Biotechnology), and actin (BD Transduction Laboratories). A standard ECL detection procedure was then used.

ALK1-Fc, ALK2-Fc and ALK3-Fc were purchased from R&D Systems. Primary antibodies for western blot analysis were purchased from Cell Signalling Technology or CalBioreagents. All peptides used in this study were purchased and synthesised by PepMic (Suzhou, China). HPAECs and endothelial growth medium were purchased from Lonza (Wokingham, Berkshire, UK). HMEC-1 cells were from the Centre for Disease Control (CDC, Atlanta, GA). All other cell culture media were purchased from Life Technologies.

Recombinant human GDF15 was mainly Chinese hamster ovary (CHO) cell line-derived (Cat# 957-GD, R&D Systems/Bio-Techne, Abingdon, UK). The two lots of GDF15 that were mostly used here were later tested for TGF-β content by R&D Systems: Lot# EHF1713081 (purchased early 2014) showed 169.7 pg TGF-β per μg GDF15 and Lot# EHF0914051 (purchased late 2014) showed 0.73 pg TGF-β per μg GDF15 and all lots sold since these measurements were done had to pass a quality control of maximum 20 pg TGF-β-content per μg GDF15 (R&D Systems, personal communication). We also performed experiments with “mammalian cell culture”-derived GDF15 (Cat# 120–28, Lot# 1111S396, Peprotech, London, UK) and E. coli-derived GDF15 (Cat# ab125769, Abcam, Cambridge, UK). Other recombinant human proteins (activin A, BMP9, ALK1-Fc, ALK5-Fc, TGFBR2-Fc, TGFBR2-isotype 2-Fc ACVR2A-Fc, endoglin-Fc, TGFBR3-Fc and M-CSF) were from R&D Systems, except IL-6 (Gibco, Invitrogen, Carlsbad, CA, USA). SB431542 was from Sigma-Aldrich (St Louis, MO, USA). TGFBR2 (Cat# AF-241-NA), pan-TGF-β (Cat# AB-100-NA), and GDF15 (Cat# MAB957) neutralizing antibodies were from R&D Systems. Protein G Sepharose 4 Fast Flow (GE Healthcare, Oslo, Norway) was used in the neutralizing antibodies experiment to pull antibodies out from cell culture media.

C2C12-BRE cells [51 (link)] were seeded at 30,000 cells/well in 96-well plates and grown for 3 days in DMEM containing 10% heat-inactivated FBS and antibiotic/antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). Cells were then washed once with DMEM containing 0.1% FBS and antibiotic/antimycotic; after aspiration of the washed, they were incubated in fresh 0.1% FBS overnight. The endoglin–GFP protein was expressed using the EGFP–N1–EndoglinCDS1 plasmid and purified with Strep-Tactin^®XT (GenScript Biotech, Leiden, The Netherlands). As the provided dilution of the protein stock (30 µg/mL) was limiting, media for the incubations were prepared using 10X M199 (Sigma-Aldrich, St. Louis, MO, USA). Dilutions of BMP9 or BMP10 (both from R&D Systems, Minneapolis, MN, USA) were incubated in the absence or presence of endoglin–GFP in M199 containing 1.5 mg/mL sodium bicarbonate, 4 mM L-glutamine, 25 mM Hepes, and 0.5% recombinant human serum albumin (Sigma-Aldrich, St. Louis, MO, USA). ALK1-Fc (R&D Systems, Minneapolis, MN, USA) was included as a positive control.