Normal rabbit immunoglobulin g

MKP5‐overexpressed mouse hepatocytes FL83B and control cells were stimulated with PA for 9 hours and crosslinked with formaldehyde at a final concentration of 1% for 10 minutes, followed by quenching with glycine. Cell lysates were fragmented by sonication and precleared with protein G Dynabeads and subsequently precipitated with anti‐phosphor activating transcription factor 2 (pATF2) antibody (Santa Cruz) or normal rabbit immunoglobulin G (Santa Cruz) overnight at 4°C. After washing and elution, crosslink reversal was done by incubating at 65°C for 8 hours. The eluted DNA was purified and analyzed by real‐time polymerase chain reaction (RT‐PCR) with primers specific to Cidea and Fsp27 promoters.

MIN6 cells were cultured with or without 10 μmol/L PIO for 48 h in a 10‐cm dish. We carried out the chromatin immunoprecipitation (ChIP) assay using an EZ‐ChIP chromatin immunoprecipitation kit (Merck Millipore, Billerica, MA, USA). After fixing with 1% formaldehyde, cells were lysed, briefly sonicated and immunoprecipitated at 4°C overnight. The following antibodies were used in the ChIP reactions: normal rabbit immunoglobulin G (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐acetyl histone H3 (Merck Millipore) and anti‐peroxisome‐proliferator activated receptor (PPAR; Santa Cruz Biotechnology). We washed the ChIP reactions, and eluted chromatin based on the manufacturer’s protocol. Chromatin was purified with PCR clean up columns (Qiagen), and PCRs were carried out with AmpliTaq Gold PCR Master Mix (Thermofisher).
The following primers, designed for mouse genes, were used.
ADM‐P1 forward: 5′‐ CAAACTTGGCAAGCACTCAG‐3′
ADM‐P1 reverse: 5′‐ AATGGGCTAGGACACACTCC‐3′
ADM‐P2 forward: 5′‐ CAAACTTGGCAAGCACTCAG‐3′
ADM‐P2 reverse: 5′‐ ACGGGTACTCCAAATGAAGG‐3′
ADM‐P3 forward: 5′‐ AAACCCCAATTTCCAATTCAG‐3′
ADM‐P3 reverse: 5′‐ GAAGGGGAACCAGAACAACTC‐3′