Pa5 23065

ChIP assay was conducted applying the SimpleChIP® enzymatic chromatin IP kits (Cell Signaling Technology, Danvers, MA, USA). Chromatin was incubated with antibodies against H3K9me3 (ab8898, Abcam), or JMJD2C (PA5-23065, Thermo Fisher Scientific), or IgG (serving as negative control, ab172730, Abcam) for immunoprecipitation. Eventually, the compound of immunoprecipitated protein and DNA was extracted and used for RT-PCR to confirm the binding site. The extraction of DNA was conducted as follow: samples were rinsed in the lysis buffer twice, and were rinsed in 1 M lysis buffer (50 mM Tris, pH 7.4, 1 M NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) for 4 times, and were resuspended in lysis buffer, followed by 45 min treatment with proteinase K at 45°C. Co-immunoprecipitated DNA was purified by QIAquick DNA purification columns (Qiagen, Germantown, MD, USA) and were eluted using 50 μL nuclease-free water. The primers of the ALKBH5 promoter were as follows: forward primer: 5’-TCTCCTTTAGGGGTCCTCGC-3’, reverse primer: 3’GGAGTTTCCGGAAGTCGGTT-5’.

The total protein was extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing 1% phenylmethanesulfonylfluoride and 1 mmol/L β-glycerophosphate sodium salt hydrate. The bicinchoninic acid protein quantification kit (Beyotime Institute of Biotechnology) was used to measure the protein concentration. After being separated by 10% SDS-polyacrylamide gel (Beyotime Institute of Biotechnology), protein samples were transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% skim milk at room temperature for 2 h, and incubated with primary antibodies against JMJD2C (PA5-23065, 1:10000, Thermo Fisher Scientific), HuR (ab200342, 1:1000, Abcam), and β-actin (ab8227, 1:1000, Abcam) at 4°C overnight and then incubated the secondary antibody (ab205718, 1:2000, Abcam) at room temperature for 1 h. Subsequently, the chemiluminescence signal were visualized using the enhanced chemiluminescence kit (Millipore). Protein levels were appraised after exposure with X-ray film (Fuji Photo film Co., Ltd, China) and were normalized to β-actin, and analyzed using ImageJ software.