Total smad5

Western blotting was carried out with following primary antibodies: Cited1 (made in our lab or Genetex, GTX114559), Oct4 (made in our lab), Sox2 (made in our lab), phospho-Smad1/5 (Cell Signaling Technology, #9516), phospho-Smad2 (Cell Signaling Technology, #3101), total Smad5 (Cell Signaling Technology, #9517), total Smad2/3 (Cell Signaling Technology, #3102), Smad4 (Proteintech, 10231–1-AP), Bmpr2 (Abcam, ab96826), Elf5 (Santa Cruz, sc-9645), T (R&D, AF2085), Placental lactogen 1 (Santa Cruz, sc-376436), Flag (Abmart, M20018F), β-Actin (HuaBio, M1210–2) and α-Tubulin (Sigma, T5168). The inhibitors were purchased from STEMCELL (SB431542: #72232), Selleckchem (LDN193189: S2618; K02288: S7359; protease inhibitor cocktail: B14002; phosphatases inhibitor: B15002) and PeproTech (Noggin: #120–10 C), respectively.

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.