2.8. Chromatin Immunoprecipitation (ChIP)

The enrichment of DNMT1, DNMT3a, and DNMT3b in the SOCS3 promoter region was assessed using a ChIP kit (Millipore). MG63 cells were incubated until cell confluence reached 70–80% and then fixed in 1% formaldehyde for 10 min to cross-link DNA and protein. Cells were randomly lysed using ultrasonic treatment to obtain 200–1,000 bp DNA fragments. After centrifugation at 4°C, the supernatant was collected and incubated with the following specific antibodies: anti-DNMT1, anti-DNMT3a, or anti-DNMT3b. Mouse IgG was used as the negative control. After decrosslinking at 65°C overnight, DNA was extracted, purified, and collected for PCR assay.