Anti rfx1 antibody

ChIP analysis was performed according to the instructions provided with the ChIP assay kit (Millipore, Billerica, MA, USA). In brief, CD14⁺ monocytes were fixed for 10 min at RT with 1% formaldehyde. Glycine was subsequently added to a final concentration of 0.125 M to quench the formaldehyde. Cells were pelleted, washed once with ice-cold PBS, and lysed with SDS buffer. Lysates were pelleted, resuspended, and sonicated to reduce DNA to fragments of 200 to 1000 base pairs. Chromatin was precipitated with protein A agarose beads for 1 h and then incubated with an anti-RFX1 antibody (Santa Cruz), anti-DNMT1 antibody (Abcam), anti-HDAC1 antibody (Abcam), anti-SUV39H1 antibody (Abcam), histone H3ac (pan-acetyl) antibody (Active motif), histone H4ac (pan-acetyl) antibody (Active motif), and histone H3 trimethyl Lys9 antibody (Active motif) or normal IgG (Millipore, Darmstadt, Germany) overnight. The immunocomplexes were further precipitated with protein A agarose beads, washed, and eluted in 100 mL of TE with 0.5% SDS and 200 mg/mL proteinase K. Precipitated DNA was further purified with phenol/chloroform extraction and ethanol. The relative enrichment level was quantified using qPCR and calculated relative to the respective input DNA [33 (link)]. The primers used are shown in Additional file 1: Table S1.

ChIP assays were implemented via ChIP Kit ab500 (Abcam, Cambridge, UK). Briefly, cells (1×10⁷) were immobilized by 1% formaldehyde. The reaction of cross-linking was ceased via glycine. A Microson XL ultrasonic cell disruptor XL (Misonix, New York) was employed to clip the chromatin. 10 μl sonicated samples were collected as input. The surplus samples were cultured with anti-RFX1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-H4K20me1 antibody (Abcam, Cambridge, UK) or IgG at 4 °C overnight. The cross-linking between DNA and protein was ceased by culture at 65 °C for 2 hours. After being purified, the occupied DNA sequences were determined by PCR. The primer sequences of ENO1 were list as below: forward, 5'-AACGCCATGGAAACCTGTCT-3', and reverse, 5'-TCTGGTCAGCTGCAAAACCT-3'.

Anti-RFX1 antibody (Santa Cruz, Clone: I-19) used for ChIP assays was purchased from Santa Cruz. H3ac (cat. no. 39139), H3K4me3 (cat. no. 39915), and H3K9me3 (cat. no. 39765) antibodies were from Active Motif. Phosphorylated STAT3 antibody (clone: D3A7) was from CST. DNMT1 (cat. no. ab13537), HDAC1 (cat. no. ab7028), and SUV39H1 (cat. no. ab8898) were from Abcam. ChIP experiments were carried out with a ChIP Assay Kit (Millipore) according to the manufacturer’s protocol. Briefly, 2 million cells were cross-linked with 1% formaldehyde, washed with cold phosphate-buffered saline, and lysed in buffer containing protease inhibitors (Roche). Cell lysates were sonicated to shear DNA and sedimented, and diluted supernatants were immunoprecipitated with the indicated antibodies. In all, 10% of the diluted supernatants were kept as “input” (input represents PCR amplification of the total sample). Real-time PCR primer sequences are shown in Supplementary Table 4. The amount of immunoprecipitated DNA was subtracted from the amount of amplified DNA, which was bound by the nonspecific normal IgG and subsequently calculated relative to the respective input DNA.