Ab54984

ChIP was performed with MagnaChIP HiSens Chromatin IP Kit (17–10461, Merck Millipore) following the manufacturer’s protocol. In brief, cells were cultured under hypoxia for 32 h and then cross-linked with formaldehyde at 37 °C for 10 min, quenched with glycine, and then sonicated to generate 300–600 bp DNA fragments using an Ultrasonic Cell Disruptor (Diagenode, Liège, Belgium). The antibodies against HIF1α (610959, BD), PKM2 (4053 S, CST), biotin-ab (033700, Invitrogen), p300 (ab54984, Abcam), H3K9ac (ab4441, Abcam), were used to for immunoprecipitation. The binding of the HIFAL promoter to HIF1α, PKM2, biotin-ab, or IgG was quantified using quantitative PCR with primers. Chip primers sequences were listed in Supplementary Table 1.

ChIP assays were performed as described previously [53 (link)]. Briefly, the cells were digested with ChIP lysis buffer (50 mM Tris-HCL PH=8.0, 5 mM EDTA, 0.1% deoxycholate, 1% Triton X-100, 150 mM NACL in 1* PIC (protease inhibitor)), And were crosslinked with 1% formaldehyde and sonicated for 180s (10s on and 10s off) on ice shear the DNA to an average fragment size of 200-1000bp. The 500ul of sonicated chromatin was purified by centrifugation, and then, the supernatants were incubated with the ChIP grade antibody against 2-5ug anti-KAT3B/P300 (Abcam, ab54984), anti-Histone H3 (acetyl K27) (Abcam, ab4729) and 100ul Dynabeads^TM protein G (Invitrogen, 10004D, USA). Finally, chromatin DNA was subjected to Quantitative PCR and all primers for ChIP-qPCR are listed in Table 1.