Anti acvr2a

Western blotting was performed as previously described.^{12 (link)} Briefly, cells were lysed in buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (cOmplete; Roche Diagnostics, Basel, Switzerland). Protein concentrations were determined using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). After samples were heat-denatured in Laemmli sample buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue), 10 μg of protein was separated in 4–15% or 10% SDS–PAGE gels and transferred onto Immobilon-P polyvinylidene difluoride membranes. After blocking for 1 hour in 5% skim milk, the membranes were probed with appropriate primary antibodies overnight at 4°C. Finally, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies for 1 hour. Signals were detected with an Enhanced Chemiluminescence Kit (Amersham Biosciences/GE Healthcare, Piscataway, NJ). Image analysis software (ImageJ, ver. 1.44; NIH, Bethesda, MD) was used to quantify band intensities, and values were normalized to β-actin levels. The antibodies used were anti-ACVR2A (#ab135634; Abcam, Cambridge, MA, USA), anti-phospho-Smad2 (#3108; Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (#A5441; Sigma–Aldrich, St. Louis, MO, USA).