Anti c ebpβ antibody

Chromatin immunoprecipitation (ChIP) assays were performed using a SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling, no. 9003, USA), according to the manufacturer's protocol. Briefly, KATO III cells were treated with 1% paraformaldehyde for protein-DNA crosslinking and then neutralized with glycine (Cell Signaling, no. 7005S, USA). Samples were treated for nuclei extraction and DNA shearing with Micrococcal Nuclease (Cell Signaling, no. 10011, USA) for 20 min at 37°C to obtain DNA fragments of approximately 150–900 bp. Nuclei were sonicated to break the nuclear membrane, chromatin was immunoprecipitated with anti-C/EBPβ antibody (Cell Signaling, no. 90081S, USA), or negative control without antibody overnight at 4°C, and then incubated for 2 h with ChIP-Grade Protein G Magnetic Beads (Cell Signaling, no. 9006, USA). DNA was eluted, de-crosslinked for 2 h at 65°C, and purified. The binding of C/EBPβ to the TFF1 promoter was assessed by PCR using the primers reported in electronic supplementary material, table S1. The results were analysed using per cent input method. Briefly, the Ct value was adjusted for the input sample (2% of total DNA used) and expressed as a percentage of the total input chromatin.

Rat neutrophil chromatin was isolated and ChIP assays were performed using a Pierce™ Magnetic ChIP Kit (Thermo, 26157) as previously described^{80 (link)}. Chromatin was immunoprecipitated using anti-C/EBPβ antibody (1:50, Cell Signaling Technology, 3082), as well as Normal Rabbit IgG (1:1000, Cell Signaling Technology, 2729) and anti-Histone H3 antibody (1:1000, clone D2B12, Cell Signaling Technology, 4620) for negative and positive controls, respectively. C/EBPβ-binding sequences were amplified with the respective gene primers listed in Supplementary Table 1.