Equal amounts of extracted proteins (20 to 40 μg) were resolved on 10% SDS–polyacrylamide gel electrophoresis gels, and the proteins were transferred to nitrocellulose membranes (Whatman). The membranes were incubated with TGFBR1 (1:1000, 3712; Cell Signaling Technology), SMAD2 (1:1000, 3102; Cell Signaling Technology), pSMAD2 Ser465/467 (1:1000, 138D4; Cell Signaling Technology), SMAD4 (1:1000, 46535; Cell Signaling Technology), and β-actin (1:5000, 3700; Cell Signaling Technology) or β-tubulin (1:5000, 2146; Cell Signaling Technology) followed by incubation with appropriate horseradish peroxidase–conjugated antibodies at room temperature for 1 hour. Proteins were detected by an enhanced chemiluminescence solution (Thermo Fisher Scientific).

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