2.12 Chromatin immunoprecipitation (ChIP) assay
1% formaldehyde was used to cross-link VSMCs for 10 min at room temperature, then glycine (0.125M) was added for 5 min. The ChIP assay was performed with Smd3 or lgG antibody (Abcam, USA) and the ChIP kit (Wanleibio, China) according to the manufacturer’s instructions. Bound DNA fragments were subjected to PCR. For PCR, 2 μL of DNA was amplified with 2×Taq PCR Master-mix (Bioteke Corp., China), using a primer pair specific to the binding region of the miR-26a promoter. The primer sequences and the condition are in Supplemental Table S1 and S2, respectively. Finally, PCR products were visualized on 2% agarose gels.