ChIP Assay for HIF-1α Binding

ChIP assays were performed, as described previously [67 (link),68 (link),69 (link)]. Cells were crosslinked with a final concentration 1% formaldehyde for 10 min at room temperature. Then, 125 mM of glycine was added to quench unreacted formaldehyde. Cells were collected and sonicated to make DNA fragments with a size range of 200 to 1000 bp. Cell extracts were immune-precipitated using 1 μg anti-HIF-1α or rabbit IgG control (Abcam, Cambridge, UK) for each sample suspended in 450 μL ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) purchased from Cell signaling Technology (Cat# 20-153, Danvers, MA, USA). For all ChIP experiments, PCR analyses were performed by using multiple sets of primers spanning the transcription factor binding site on the NPHP3 gene promoter.