Anti notch1 antibody

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).

For immunoblot analysis, isolated colon crypts or Caco-2/BBe cells scraped off from the dish were lysed with NP40 lysis buffer (50 mmol/L TrisHCl, 150 mmol/L NaCl, 1% NP40, pH 8.0) supplied with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) on ice, we used 1 ml syringe to pass the lysates through 27 Gauge needle 6 times and samples were kept on ice for 20 min lysis before 5 min 6000 rpm centrifugation at 4 °C. Supernatants were collected for SDS-PAGE analysis. The following primary antibodies were used: anti- CLDN1 antibody (1:10000; 51–9000; Thermo Fisher, Waltham, MA, US), anti HES1 antibody (1:10000; sc-25392, Santa Cruz, Dallas, TX, US), anti- Notch1 antibody (1:2000; #3608; Cell Signaling Technology, Danvers, MA, US), anti- Cleaved Notch1 antibody (1:2000; #4147; Cell Signaling Technology, Danvers, MA, US), anti- NR3C1 antibody (1:10000; #3660; Cell Signaling Technology, Danvers, MA, US), anti-β-actin antibody (1:20000; A1978, Sigma-Aldrich, St. Louis, MO, US) overnight at 4 °C. The corresponding immunoblot bands were scanned at 1200 dots per inch and analyzed with ImageJ.