Smoc1

About 4-μm paraffin-embedded tissue sections were used for immunohistochemistry staining. Secondary antibody (Cat. 111-035-003; Jackson) conjugated with horseradish peroxidase (HRP) was used after the overnight incubation of the slides with the primary antibody SMOC1 (Cat. ab155776, Abcam) at 4°C. Next, the slides were incubated in DAB solution for 3 min and counterstained with haematoxylin for 5 min. Digital images were acquired using a microscope (Axio Imager.A2; Carl Zeiss).

For immunoprecipitation, the samples were collected and lysed in 50 mmol/L of Tris-HCl (pH 7.4) lysis buffer. The supernatant was immunoprecipitated with 5 μg of anti-BMPR-II (Cat. sc-393304; Santa Cruz, USA), anti-FLAG (Cat. F3165; Sigma), anti-Myc (Cat. 05-724; Millipore, USA) or anti-SMOC1 (Cat. ab155776; Abcam) overnight accordingly and then was incubated with Dynabeads™ Protein A (Cat. 1002 D, Invitrogen) for another 3 h at 4°C. Next, the cell lysates were subjected to western blot analysis as described previously.² (link) The primary antibodies used were SMOC1 (Cat. ab155776; Abcam), RUNX2 (Cat. 12556; Cell Signaling Technology), BMP2 (Cat. ab14933; Abcam), p-Smad1/5 (Cat. 9516; Cell Signaling Technology), Smad1 (Cat. 6944; Cell Signaling Technology), p-p38 (Cat. 4511; Cell Signaling Technology), p38 (Cat. 9212; Cell Signaling Technology), Caspase-3 (Cat. 9662; Cell Signaling Technology), BMPR-II (Cat. sc-393304; Santa Cruz), FLAG (Cat. F3165; Sigma) and Myc (Cat. 05-724; Millipore). Image Lab™ software was used to quantify the band density.