ChIP assays were performed as previously described [23 (link)]. Briefly, glycine was added to the cells to a concentration of 0.125 M to quench the crosslinking. The cells were then rinsed with ice-cold PBS, re-suspended, lysed in lysis buffer, and sonicated to shear the crosslinked DNA to fragments ranging from 200 to 500 bp, as previously described [24 (link)]. The lysates ere incubated with specific antibodies or normal IgG at 4 °C overnight. After adding 40 μL of protein G magnetic beads, the lysates were further incubated for 2–3 hours. The beads were washed repeatedly, and the DNA was eluted from the beads by incubating in 10 mM Tris-Cl, pH 8.5, for 15 minutes at 65 °C. Both the immunoprecipitated and the input DNA samples were incubated overnight at 65 °C for reversal of the crosslinking. The DNA samples were then purified by sequential phenol/chloroform/isoamyl alcohol (Sigma) extraction. The final DNA products were ethanol-precipitated, and the pellets were air-dried and dissolved in 10 mM Tris-HCl. The primers were used for the ChIP assay as previously described [25 (link)]. Anti-RBPJκ (Abcam, ab25949), anti-NICD1 (Abcam, ab83232), and anti-MAM (Abcam, ab17019) antibodies were used for the ChIP assays.
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