Ecl western substrate

Total protein was isolated using Complete LysisM Buffer (Roche, Indianapolis, IN, USA). Protein was diluted to equal concentrations and equally loaded on 10% polyacrylamide gels prior to transfer to a nitrocellulose membrane. Protein concentration was determined using micro plate BCA assay (BioRad, Hercules, CA, USA). Blots were incubated overnight with Smad1 (Cell Signaling Cat#6944 1:1000), Smad 2 (Cell Signaling Cat#5339 1:1000), pSmad1/5 (Cell Signaling Cat#9516 1:1000), pSmad2 (Cell Signaling Cat#3108 1:1000), and Actin (Sigma Cat#A2066 1:4000) antibodies. HRP-conjugated secondary antibodies were used to visualize band intensity via x-ray film exposure using ECL western substrate (Perkin Elmer, Waltham, MA, USA). Conditioned medium was collected 48 h after equal cell numbers were plated into T-75 culture flasks. Supernatant was spun to remove cells and debris and 500 μL were used per membrane for Human cytokine array XL, performed following manufacturer instructions (RnD Systems Cat#ARY006) with assistance from VAPR (Vanderbilt Antibody Protein Resource). sTβRIII was no longer present in the conditioned medium collected, and was replaced with new medium after 48 h of treatment followed by an additional 48 h of medium conditioning, which was then collected. We did not alter the serum level or perform starvation.