Western Blot Analysis of EMT and BMP Signaling

Western blot analysis was performed according to a previously described standard method (37 (link)). The primary antibodies used for the Western blot analyses were as follows: 14-3-3ζ (Santa Cruz Biotechnology, SC-293415, diluted at 1:500), E-cadherin (Abcam, ab133597, diluted at 1:5,000), N-cadherin (Abcam, ab76057, diluted at 1:1,000), vimentin (Abcam, ab137321, diluted at 1:1,500), BMP2 (Abcam, ab214821, diluted at 1:1,000), BMPR2 (Abcam, ab124463, diluted at 1:500), Smad1 (Cell Signaling Technology, #6944, diluted at 1:1,000), Smad5 (Cell Signaling Technology, #9517, diluted at 1:1,000), phosphorylated (p)-Smad1/5 (Cell Signaling Technology, #9516, diluted at 1:1,000), ID1 (Santa Cruz Biotechnology, SC-374287, diluted at 1:500) and β-actin (used as the loading control; Sigma, A1978). Western blot bands were quantified by ImageJ software (U.S. National Institutes of Health, USA). The experiments were repeated three times.