Ab83232

The dissected trunk regions of zebrafish embryos were collected for extracting protein. Western blotting was performed^{60 (link)} with 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred into a nitrocellulose membrane and then membranes were blocked by nonfat milk. After blocking, the nitrocellulose membranes were then incubated using anti-Fsd1 antibody (Ab) (1:200, monoclonal mouse or polyclonal rabbit anti-FSD1 antibody was generated using a glutathione-S-transferase fusion protein containing the full-length FSD1, which were expressed in E. coli and then were purified to homogeneity as the antigen. The monoclonal mouse Ab was used in human RPE-1 cells, while the polyclonal rabbit Ab was used in zebrafish experiments), anti-Runx1 Ab (1:200, AS-55593, Ana Spec), anti-NICD Ab (1:400, ab83232, Abcam, used in zebrafish), anti-NICD Ab (1:1000, ab8925, Abcam, used in human cells), mouse anti-β-actin (1:1000, sc47778, Santa Cruz Biotechnology, used in human cells), and anti-β-actin Ab (1:2000, 4967, Cell Signaling). The figures for western blotting were collected by Imagequant LAS 4000 (The uncropped scans of blots are listed in the Supplementary Figure 12).

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.

WB experiments were performed as previously reported [69 (link),93 (link)] using the following antibodies: anti-β-Actin antibody (4967; Cell Signaling Technology, Danvers, Massachusetts), anti-NICD antibody (ab83232; Abcam, Cambridge, UK), anti-Myc antibody (06–549; Millipore), anti-Flag antibody (F7425; Sigma-Aldrich), anti-DLL4 antibody (ab7280; Abcam), anti-JAG1 antibody (ab7771; Abcam), anti-Notch1 antibody (3608; Cell Signaling Technology), anti-Erk antibody (9102; Cell Signaling Technology), anti-p-Erk antibody (9101; Cell Signaling Technology), anti-p-AKT antibody (4060; Cell Signaling Technology), and anti-AKT antibody (9272; Cell Signaling Technology). Quantification of protein level using gray analysis (Gel-Pro analyzer). Immunofluorescence experiments were performed as previously reported [94 (link)] using the following antibodies: anti-Notch1 antibody (3608; Cell Signaling Technology), anti-Notch1 antibody (3447; Cell Signaling Technology), anti-DLL4 antibody (ab7280; Abcam), anti-JAG1 antibody (ab7771; Abcam), anti-LAMP1 antibody (15665; Cell Signaling Technology), anti-EEA1 antibody (610457; BD Biosciences, Franklin Lakes, New Jersey), anti-APPL1 antibody (3858; Cell Signaling Technology), anti-AKT antibody (2920; Cell Signaling Technology), anti-AKT antibody (9272; Cell Signaling Technology), and anti-PIK3CA antibody (ab40776; Abcam).