Epiquik chromatin immunoprecipitation kit

A ChIP assay was performed using an EpiQuik TM Chromatin Immunoprecipitation Kit (Epigentek) following the protocol provided by the supplier. 2 × 10⁶ Jurkat-HBZ cells and control cells were used in each assay. The sonicated samples were immunoprecipitated with the NRF-1 antibody (ab34682, Abcam) or normal rabbit IgG (sc-2027 X, Santa Cruz) overnight at 4 °C. Protein-DNA complexes were de-crosslinked at 65 °C for 6 h. Primers used for the quantification of the target fragments by real-time PCR were 5′-TGCCGCCAGGGTTTGAA-3′ and 5′-CTGAGGCGCACAGAACCAAC-3′, which were constructed to contain the NRF-1-binding sequence located in −41/−30 bp of the TDP1-gene promoter. Individual PCRs were carried out in triplicate, and mean Ct values were collected.

The assay was performed using the EpiquikTM Chromatin Immunoprecipitation Kit (Epigentek) according to the instructions. H9C2 cells were incubated with 1% formaldehyde for 10 min, glycine was used to terminate cross-linking, and then 400 μL SDS lysis solution containing a protease inhibitor. Cells underwent ultrasound crushing and centrifugation to collect the supernatant. ChIP dilution buffer, 20 μL 50× IPC, and 60 μL Protein A Agarose/salmon sperm DNA mixture were added to 100 μL of the supernatant, mixed at 4° C for 3 h, and centrifuged to obtain the supernatant. Monoclonal antibodies (2 μL) against acetylated H3 were added to the supernatant and incubated overnight at 4° C. The precipitate was collected by centrifugation and washed with solutions in the following order: TSI, TSII, Buffer III, and TE solutions. The eluent was then added and the supernatant was collected by centrifugation. NaCl (5 mol/L) was used to unlock the crosslinking, and the DNA was extracted and recovered. PCR was performed using Nrf2 promoter region-specific primers: 5'-AGGGTCACAGCATTAGG-3' (sense); 5'-ACAGGGTTCCTTTCCAT-3' (antisense).

The ChIP assay was performed using the EpiQuikTM Chromatin Immunoprecipitation Kit from Epigentek Group Inc. (Brooklyn, NY). Protein–DNA complexes were immunoprecipitated with HBx, survivin or Sp1, with Anti-RNA polymerase II as a positive control antibody and normal mouse IgG as a negative control antibody. PCR amplification was performed using 1 μl of each DNA sample. Amplification of soluble chromatin prior to immunoprecipitation was used as an input control.

ChIP assay was conducted using an EpiQuik^TM chromatin immunoprecipitation kit (EpiGentek, Farmingdale, NY, USA, P-2002) following the manufacturer’s instructions. Samples including protein-chromatin complexes were incubated with ChIP grade antibody, the RNA polymerase (RNAPol), and the normal IgG. RNAPol and normal IgG were used as a positive control and negative control, respectively. Sample DNA was acquired by supplied column and amplified by PCR using a designed primer (Table S2). One percent of the sample chromatin extract was used as an input.

In vivo binding of ICP4 to the promoter of has-mir-101-2 was investigated using the ChIP assay according to the protocol of EpiQuik^TM Chromatin Immunoprecipitation Kit (Epigentek Group Inc, USA). Briefly, HeLa cells were transfected with pICP4-FLAG. After 24 h, cells were trypsinized and collected, after washed by PBS followed by treated with 1% formaldehyde for 10 min at 37 °C and washed twice in ice-cold PBS. Samples were sonicated to shear DNA to lengths between 200 and 1000 bp. Subsequently, an aliquot of sheared DNA was analyzed by agarose gel electrophoresis to confirm sheared DNA length and standardize protein-DNA complex input for immunoprecipitation. The chromatin samples were incubated overnight at room temprature with mouse anti-FLAG monoclonal antibody (MBL, Japan). DNAs were purified by EpiQuik^TM Chromatin Immunoprecipitation Kit. PCR was performed with primers flanking the predicted ICP4 binding site in the promoter sequence. All of the primers for PCR amplification are illustrated in Table S1.

EpiQuikTM Chromatin Immunoprecipitation Kit (Epigentek, Farmingdale, NY, USA) was used to measure the DNA binding level of NF-κB p65 with MCP-1 promoter according to the instructions. The amplified sequences of MCP-1 primer were: forward, 5′-CCAGCCAAATGCATTCTCTTCTA-3′; reverse, 5′-GAGGTCAGTGCTGGCGTGA-3′. 1% paraformaldehyde was used to fix at room temperature for 10 minutes after washing the cells with PBS, and cell lysis buffer was used to scrape the cells after washing the cells with PBS again. Then, ultrasonic was used to shear chromatin (20–25% of the output power was used), 3-4 times for 10–12 seconds. The cells were placed on ice for 30-40 seconds between every 10–12 second session. If necessary, 5 μl ultrasonic cell lysis buffer was taken for agarose gel electrophoresis, and the length of the shear DNA fragments was usually 200–1000 bp. Lysates were cleared by centrifugation (16,000 × g) at 4 °C for 10 minutes, and the supernatant was taken. According to instructions, 10 μl of NF-κB p65 antibody was added to bond to the assay plate, and after the protein/DNA immunoprecipitation, reverse of crosslinked DNA, DNA purification and PCR amplification, the final PCR product was analyzed by 1.5% agarose gel electrophoresis.