Nbp1 30873

ChIP assays were implemented with a Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, MA) according to the manufacturer’s instructions. In brief, HUVECs were fixed with 1% formaldehyde for 10 min at room temperature to mediate DNA and protein cross-linking. Glycine was then used to terminate the DNA and protein cross-linking reaction. Chromatin was sheared with the use of a Microson Ultrasonic Cell Disruptor XL (Misonix). Ten microliters of the sonicated solution was collected from each sample as input, and the remaining sample was incubated with anti-ELF3 (NOVUS, NBP1-30873), and anti-histone H4 lysine 20 methylation (H4K20me1; Abcam, ab9051) antibodies or a negative control IgG at 4 °C overnight. Immunoprecipitants were bound to protein G magnetic beads, and the DNA-protein cross link was reversed by incubating at 65°C for 2 h. Then, the DNA was purified, and enriched DNA sequences were analysed by qPCR. MARK4 oligonucleotide sequences for PCR primers were as follows: forward 5′-CCAACTGGGGAGAGAATGGG-3′ and reverse 5′-AGACTGAGAGAGACCCCAC-3′.

ChIP was performed as reported previously [40 (link)] with the following minor modifications. NMuMG cells were grown and treated in 10 cm plates. Following 10 min cross-linking and lysis, cell lysates were sonicated in the S220 focused-ultrasonicator (Covaris, Woburn, MA, USA) for 30 min (60 s on/60 s off; Peak Power: 140 W; Duty factor: 15; Cycles/burst: 200; water temperature: 4 °C). Immunoprecipitation was performed at 4 °C for 16 h using an antibody against ELF3/ESE-1 (NBP1-30873, Novus Biologicals, Centennial, CO, USA) and a rabbit control IgG (2729S, Cell Signaling, Danvers, MA, USA). Quantitative real-time PCR was performed using the TaqMan Universal master mix II and Universal Probe Library probes (Roche, Basel, Switzerland) and amplification was carried out in the ABI PRISM 7500 Fast qPCR System (Thermo Scientific, Waltham, MA USA) with primers and probes depicted in Table S5 at 95 °C for 15 min, 50 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were normalized to the input and presented as percentage of input.