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.