Chromatin immunoprecipitation (ChIP) analysis

The cultured MIN6 cells were infected with adenovirus encoding NR4A1-HA or control adenovirus. After 48 h post-infection, the infected cells were applied for ChIP assay. ChIP analysis was accomplished with a ChIP Assay kit (Beyotime). The experiments were carried out according to the manufacturer’s instructions. During this process, the genomic DNA was broken up to 200–1000 bp fragments by sonication. The antibody applied for HA-tagged NR4A1 was an anti-HA monoclonal antibody (12CA5), purchased from Roche. The pull-down DNA products were used for PCR analysis with specific primers. Agarose gel electrophoresis was used to detect if NRA1 could associate with particular promoter regulatory elements in genomic DNA.

The following two pairs of primers were, respectively, designed to amplify the specific targeting sequences of −86 bp to −17 bp and –1663 bp to −1534 bp in the MKP7 regulatory region.

5′-GGATTGGTTTCAAGTGACGCCATCTC-3′ (F),

and 5′-GAGAAAGAGTCGCTGGTCAGGAAACTTC-3′ (R);

5′-GGGCTCCCTTTTTCACTCTGAAAGATGACC-3′ (F),

and 5′-GTCCTCTCCCACCTTTAAACCCTGCAAC-3′ (R)