Ab150404

C666-1 cells were fixed and cross-linked with 1% formaldehyde, collected with PBS, and resuspended in a lysis buffer. Genomic DNA was fragmented by a Covaris E220 ultrasonicator to approximately 200–500 bp, and then the supernatants were diluted and shaken overnight with an anti-ZIC2 antibody (ab150404, Abcam) at 4 °C. The next day, protein A/G beads were added and incubated at 4 °C for 1 h. After repeated washing of the immune complexes, the eluted DNA was uncrosslinked with 5 M NaCl in a 65 °C water bath for 4 h, and DNA was purified with the QIAquick PCR Purification Kit (QIAGEN, #28104, Hilden, Germany). Purified NDA was used for ChIP-seq or real-time PCR. The sequences of the primers used for ChIP-qPCR were as follows:
JUNB −1297 gDNA-F: 5'-CAACACCGTGTCGGCTCCTA-3';
JUNB −1297 gDNA-R: 5'-CACGCCCAGGTTCCTCTTCC-3';
JUNB −123 gDNA-F: 5'-CGTGGCCGCTGTTTACAAG-3';
JUNB −123 gDNA-R: 5'-TTTCCTGGCGTCGTTTCC-3';
JUNB +2163 gDNA-F: 5'-CCCATACAAGGACCGATTCTGC-3';
JUNB +2163 gDNA-R: 5'-GCGCCAGTGTCTTGAAGGTG-3';
JUNB +2676 gDNA-F: 5'-TGTCTGTTCCACTCACCCTA-3';
JUNB +2676 gDNA-R: 5'-CCTGCCAGTTTCCCCTCA-3';
JUNB +4740 gDNA-F: 5'-CAGAGTCCCTGCTGTGAG-3';
JUNB +4740 gDNA-R: 5'-CTGTAGGAGGAAATTGGG-3';
GAPDH-gDNA-F: 5'-CCCCACACACATGCACTTACC-3'; and
GAPDH-gDNA-R: 5'-CCTAGTCCCAGGGCTTTGATT-3'.

Total protein was obtained with sodium dodecyl sulfate (SDS) sample buffer, and the protein was separated by 9% SDS-PAGE. Then, the protein was transferred to a PVDF membrane (Millipore, Burlington, MA). Membranes were blocked in 5% milk at room temperature for 1 h and subsequently incubated with an anti-ZIC2 antibody (ab150404, Abcam, Cambridge, UK) or anti-JUNB antibody (3753 S, CST, Danvers, USA) overnight at 4 °C. GAPDH (6004-1, Proteintech, Wuhan, China) and β-actin (66009-1-1g, Proteintech) served as internal controls. Species-matched secondary antibodies were then incubated with the PVDF membranes at room temperature for 1 h. Finally, enhanced chemiluminescence (Thermo, Waltham, MA) was used to visualize the antigen-antibody reactions.