Runx2

Primary antibodies include VEGF (Cat# sc‐80436, RRID:AB_1131207, Santa Cruz Biotechnology), OPN (Cat# sc‐10593, RRID:AB_2270967, Santa Cruz Biotechnology), ALP (Cat# sc‐365765, RRID:AB_10842161, Santa Cruz Biotechnology), OCN (Cat# ab13418, Abcam), Runx2 (Cat# MBS186093, RRID:AB_10888180, MyBioSource), TGF‐β3 (Cat# ab90264, RRID:AB_2050388, Abcam), Smad3 (Cat#NBP1‐88732, RRID:AB_11034477, Novus), Wnt3 (Cat# ab32249, Abcam), β‐catenin (Cat# 26170, RRID:AB_2629235, NewEast Biosciences) and GAPDH (Cat# JM‐3777‐100, RRID:AB_843142, MBL International). LNA anti–miR‐29b oligonucleotides (antagomiR‐29b) and control LNA–scrambled oligonucleotides were custom generated and provided by GenePharma. As a negative control (NC), 10 point mutations were made in the miR‐29b mature sequence generating an RNA sequence that is not expressed in the rat genome.

The total proteins in the osteoblasts were extracted and the protein concentrations were determined by the BCA protein assay kit (Beyotime, Shanghai, China). 30 μg of the total proteins were subjected to 10% SDS-PAGE and then transferred onto PVDF membranes. After being blocked in TBS containing 5% skimmed milk for 1 h, the membranes were co-incubated with the primary antibodies at 4 °C overnight against RUNX2 (1:1000 dilution; MyBioSource, cat.no.127554, San Diego, CA, USA), OPN (1:1000 dilution; Proteintech, cat.no.22952-1-AP, Rosemont, IL, USA), GLP-1R (1:1000 dilution; ABGENT, cat.no.AP52040), ABCA1 (1:1000 dilution; Affinity, cat.no.DF8233), apoA-I (1:1000 dilution; Affinity, cat.no.DF6264), and GAPDH (1:1000 dilution; Affinity, cat.no.AF7021), and then with HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution; Boster Biological Technology, Wuhan, China). Protein bands were detected using the enhanced chemiluminescence detection system and quantified using the Fiji ImageJ (version 1.51r; NIH, Bethesda, MD, USA).