Ab213223

The AMI and CAD tissues were lysed using strong RIPA buffer containing Halt Protease Inhibitor Cocktails (Thermo Fisher Scientific, Waltham, USA). Protein concentrations were evaluated with a bicinchoninic acid assay kit (Beyotime, Nantong, China). Primary antibodies targeting to beta actin (ab8226, Abcam), UHRF2 (ABE1028, MilliporeSigma), TET3 (ab153724, Abcam), UHRF2 (ZBTB33, MilliporeSigma), TET1 (ab19198, Abcam), DNMT3B (ab2851, Abcam), NTHL1 (ab191413, Abcam), UHRF1 (ab213223, Abcam), MBD3 (ab157464, Abcam), and SMUG1 (ab192240, Abcam), were incubated with targeted proteins at 4°C overnight, followed by incubating with appropriate horseradish peroxidase-conjugated secondary antibodies. Detection of horseradish peroxidase was performed with the Super Signal West Pico Chemiluminescent Substrate (Pierce).

Total proteins were extracted from cultured cells and the protein concentration was measured by BCA kit (Beyotime). Equal amounts of protein lysates were separated on a 10% SDS-PAGE, transferred to PVDF membrane and then immunoblotted with antibodies against Geminin (1:1000 dilution; Cell Signaling Technology, #5165, Danvers, U.S.A.), UHRF1 (1:1000 dilution; Abcam, ab213223, Cambridge, U.K.) and β-actin (1:5000 dilution; Absci, AS014, Nanjing, China), followed by incubating with HRP goat anti-rabbit IgG (ABclonal, Wuhan China). As for UHRF1 and Geminin, the dilution of the HRP goat anti-rabbit IgG antibody was 1:2000; as for β-actin, the dilution was 1:10,000. The primary antibody UHRF1, Geminin and β-actin were incubated at 4°C overnight. The second-antibody HRP goat anti-rabbit IgG was incubated at room temperature for 2 h. The results were visualized using Image Lab Imaging System software.