Bmpr2

Western blot analysis was performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in the RIPA buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were probed with the specific primary antibodies, followed by peroxidase-conjugated secondary antibodies. The bands were visualized by chemiluminescence (Denville Scientific). The following antibodies were used: antibodies to E-cadherin (1∶1000, BD Transduction Laboratories, 610182), vimentin (1∶2000, NeoMarkers, MS-129-P), Erbb2 (1∶500, Cell signaling Technology, 2242), HOXA1 (1∶1000, Santa Cruz Biotechnology, sc-17146), SMARCA5 (1∶500, sc-8760 from Santa Cruz Biotechnology and ab3749 from Abcam), SMARCD1 (1∶500, Abcam, ab86029), mTOR (1∶1000, Cell signaling Technology, 2972), BMPR2 (1∶1000, Cell signaling Technology, 69679), cyclin D1 (1∶1000, Cell signaling Technology, 2922), ALDH1A1 (1∶1000, Santa Cruz Biotechnology, sc-22589), HSP90 (1∶3000, BD Transduction Laboratories, 610419) and cyclophilin B (1∶2000, Thermo, PA1-027A).

The following antibodies were utilized in this study: Cell Signaling Technology: BMPR2 (6979), p-Smad1/5/8 (9511), ID2 (3431), c-Abl (2862), KAT5 (12058), Flag (2368), pSTAT1 (9171), pSTAT3 (9131), and pSmad2/3 (8828). PeproTech Inc.: BMP2 (500-P195). Santa Cruz Biotechnology Inc.: BMP4 (sc-12721) and p-Tyrosine (PY99). Calbiochem: PCNA (NA-03). Abcam: TLR3 (ab62566), TICAM1 (ab13810), BMPR1a (ab38560), pPDGFR (ab5443), and p-c-KIT (ab5616). Sigma: iNOS (N7782) and β-ACTIN (A3854). Jackson Immuno Research: Goat anti-Rabbit HRP (111-035-045) and Mouse anti-Rabbit Light Chain specific HRP (211-032-171), DyLight 488 IgG Fraction Monoclonal Mouse Anti-Rabbit. Invitrogen: p-Threonine (PT-5H5). Tonbo Biosciences: PE Anti-Mouse F4/80 Antigen (BM8.1) (50-4801), APC Anti-Mouse Ly-6G (1A8) (20-1276). BD Biosciences: PE Anti-Mouse CD11c (HL3) (557401).

Cells were rinsed twice with cold PBS and lysed by RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (Roche). Protein (40 μg per sample) was separated by SDS-PAGE using a 10% polyacrylamide gel. The proteins were transferred electrophoretically onto a PVDF membrane. Blotted membranes were blocked in 5% skimmed milk diluted in TBST, followed by incubation with appropriate primary antibodies (anti-cyclin D1, CDK4, p18, Rb, p-Rb, caspase-9, caspase-3, PARP, E-cadherin, N-cadherin, Vimentin, SNAI1, SLUG, BMPR1, BMPR2, SMAD4, pSMAD1, 5, 8, Atg5, Atg7, Beclin 1, LC3, and β-actin; obtained from Cell Signaling Technology and all the antibodies were diluted 1:1000.) overnight at 4°C. The membranes were then washed for 5 minutes for three times with TBST, and subsequently incubated for 1 hour with HRP-linked secondary antibody (Cell Signaling Technology) at room temperature. β-actin was used as an internal control. The blots were detected using an enhanced chemiluminescence kit (Millipore) and subjected to autoradiography using X-ray film.