Mouse anti hes1

Samples were lysed and analysed by western blotting to determine the expression levels of the indicated proteins. The following antibodies were used. Mouse anti‐c‐MYC (1:1000), mouse anti‐HES1 (1:1000) and mouse anti‐Dishevelled (1:1000) were from Santa Cruz Biotechnology. Rabbit anti‐p‐SMAD1/5 (1:1000), rabbit anti‐β‐catenin (1:1000) and rabbit anti‐GAPDH (1:1000) were from Cell Signalling Technology. Rabbit anti‐LRP6 (1:1000 for western blot and 1:200 for immunostaining) and mouse anti‐LGR5 (1:1000 for western blot and 1:200 for immunostaining) antibodies were purchased from Immunoway. Mouse anti‐clathrin (1:200 for immunostaining) and mouse anti‐caveolin (1:200 for immunostaining) were purchased from Proteintech.

Tissues were homogenized in RIPA plus buffer containing a cocktail of EDTA-free protease inhibitors. The homogenate was centrifuged at 12,000 rpm for 30 min at 4°C. Protein concentration was assayed using the BCA method, then loaded and subjected to electrophoresis in 10% SDS-PAGE gels, and transferred onto PVDF membranes. The membranes were then blocked in 5% BSA for 2 hour and then incubated with one of the following primary antibodies: rabbit anti-caspase-3 (1 : 500, Proteintech) or mouse anti-Bax (1 : 100, Proteintech) or rabbit anti-Bcl-2 (1 : 500, Proteintech) or mouse anti-Notch1(1 : 500, Santa Cruz) or mouse anti-Hes1 (1 : 1000, Santa Cruz), with gentle shaking at 4°C overnight. Then, horseradish peroxidase conjugated goat anti-mouse IgG (1 : 50000, Proteintech) or goat anti-rabbit IgG (1 : 50000, Proteintech) secondary antibodies were incubated with the membranes for 2 hours at room temperature. The immunopositive bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher) and Bio-Rad ChemiDoc XRS digital documentation system. The amount of protein expression is presented relative to the levels of β-actin.