Crushed snap-frozen tissue samples and cell pellets were lysed in RIPA buffer (150 mM NaCl, 0.5% Na-deoxycholate, 1% Triton X-100, 0.5% SDS, 50 mM Tris-HCl (pH 7.5)) and protease/phosphatase inhibitor cocktails (catalog no. 16829900; Sigma-Aldrich). Protein lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific; catalog no. 23225). Laemmli buffer (0.1% 2-mercaptoethanol, 0.0005% bromophenol blue, 10% glycerol, 2% SDS in 63 mM Tris-HCl (pH 6.8)) was added to 20 μg of protein extracts that were then boiled for 5 min and loaded on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes at 100 V for 2h. After blocking in 5% skim milk, membranes were incubated (4 °C, overnight) with antibodies against TGFBI (1:500; Cell Signaling; catalog no. 2719), SMAD2/3 (1:1000; Cell Signaling; catalog no. 8685,) and beta actin, used as loading control (1:10000; Cell Signaling; catalog no. 4967).

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