Western Blot and Immunofluorescence Protocols

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).