Notch1 d1e11

Lysates were extracted from the heads of E12.5 embryos, MEFs, and adult livers in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS) supplemented with a Protease Inhibitor Cocktail Set III (Calbiochem). Immunoblot analysis was performed using the following primary antibodies: mTOR (7C10, Cell Signaling), Notch1 (D1E11, Cell Signaling), NF-κB2 (18D10, Cell Signaling), Myc (Y69, Abcam), Klf5 (ab24311, Abcam and G-7, Santa Cruz), c-Jun (H-79 and G-4, Santa Cruz), TGIF (H-172, Santa Cruz), Cyclin E (C19, Delta Biolabs), SREBP1 (2A4, Novus Biologicals and H-160, Santa Cruz), and β-actin (AC-15, Sigma). Each primary antibody was used at a 1:1000 dilution. Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (GE Healthcare) was used as the secondary antibody. Immunoreactive proteins were visualised using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions.

For protein extraction, adherent cells were washed with cold PBS and directly treated with cell lysis buffer (Cell Signaling, Danvers, MA, USA), freshly supplemented with 1 mM PMSF (Carl Roth, Karlsruhe, Germany) and a protease inhibitor cocktail (Cell Signaling, Danvers, MA, USA). Cells were scraped off the culture dish, incubated on ice, and centrifuged for 15 min. The Bradford assay, SDS-Page, semi dry transfer onto a polyvinylidene difluoride (PVDF) membrane, blocking, and incubation with the primary antibody were performed as described by Koch [16 (link)]. Primary antibodies against NOTCH1 (1:1000, NOTCH1 D1E11, Cell Signaling Technology, Inc., Danvers, MA, USA) or β-Tubulin antibody (1:1000, 2146, Cell Signaling Technology, Inc., Danvers, MA, USA) were used. After incubation, the membranes were washed in TBS-T and Anti-Rabbit IgG HRP-linked antibody (1:5000, Cell Signaling Technology, Inc., Danvers, MA, USA) was applied for another hour at 4 °C. The membranes were incubated with Thermo Scientific™ Pierce™ ECL Western Blotting Substrate (Fisher Scientific, Waltham, MA, USA) and chemiluminescence detection was performed with the ChemiDoc XRS+ Imager (Bio-Rad Laboratories GmbH, Munich, Germany). Analysis and editing were performed with Image Lab 6 (Bio-Rad Laboratories GmbH, Munich, Germany).