Ma5 15827

Human and mouse SMCs were harvested and lysed in a lysis buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS. Equal amounts of protein from each cell-type preparation were separated on a SDS–PAGE, blotted, and detected with rabbit anti-ALP (1:1000, NBP2-67295, Novus Biologicals, Littleton, CO), goat anti-BMPR-1A (1:1000, PA5-18494, Thermo Fisher Scientific), mouse anti-BMPR-1B (1:1000, MAB505-100, R&D Systems), mouse anti-BMPR-2 (1:1000, MA5-15827, Thermo Fisher Scientific), mouse anti-collagen I (1:1000, PA5-29569, Thermo Fisher Scientific), rabbit anti-osteocalcin (1:1000, AB10911, Sigma-Aldrich), mouse anti-osteopontin (1:1000, 691302, BioLegend), rat anti-Runx2 (1:1000, 692802, BioLegend), rat anti-TGFBR-1 (1:1000, MAB5871, R&D Systems), and rat anti-TGFBR2 (1:1000, MAB532, R&D Systems), and GAPDH (1:2000, 2118S, Cell Signaling Technology) antibodies. Western blot signals were assessed by densitometric analysis using the Image J software.

RIPA lysis buffer (89900, Thermofisher scientific, Breda, The Netherlands) with protease inhibitors (PMSF, Thermofisher scientific/complete mini protease inhibitor cocktail, Sigma-Aldrich, Zwijndrecht, The Netherlands) were added to the lung tissue and homogenized with a tissue lyser (Qiagen, Venlo, The Netherlands). Protein concentration was quantified by protein assay kit (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA). The samples (10 µg) were separated on a gradient gel (Bis-Tris 4–12% gel, Life technologies). XCell blot module was used for protein transfer from the gel to an ECL membrane (Hybond ECL Nitrocellulose Membrane, GE Healthcare). Primary antibody for BMPR2 (1:1000, MA5-15827, Thermo scientific, Breda, The Netherlands)/phospho-Smad 1/5/8 (#12656, Cell Signaling, Leiden, The Netherlands)) diluted in phosphate buffered saline with bovine serum albumin (5%) (PBS-A) was incubated overnight at 4 °C. Appropriate secondary antibody (1:4000, Polyclonal rabbit anti-mouse, Z0259, DAKO) diluted in PBS-A was incubated for one hour. The blots were re-incubated with β-actin (1:50000, A3854, Sigma) or GAPDH diluted in PBS for one hour to correct for unequal loading. An internal control composed of a mix of protein supernatants from all groups was used to correct for inter-blot variation.

Gel electrophoresis was run with NuPAGE 4–12% Bis–Tris pre-cast gels and the accompanying buffers (Invitrogen) following the manufacturer’s instructions. Antibodies against BMPR2 (1:2000, Ma5-15827, Thermo Fisher Scientific), pSMAD1/5/9 (1:1000, 13820, Cell Signaling), pSMAD2 (1:1000, gift from Prof. ten Dijke at LUMC Leiden), and GAPDH (1:10000, g9295, Sigma-Aldrich) were used for protein detection.