Rabbit anti notch

Proteins in cell culture homogenates were separated by 12% SDS-PAGE and transferred onto PVDF membranes (Amersham). Primary antibodies used in immunoblot experiments were as follows: rabbit anti-SOX2 (1 : 10000, Genetex), rabbit anti-βIII tubulin (1 : 3000, Epitomics), rabbit anti-Ascl (1 : 1000, Abcam), rabbit anti-Notch (1 : 1000, Abcam), mouse anti-p27 (1 : 6000, BD Biosciences), rabbit anti-α-tubulin (1 : 150000, Abcam), and rabbit anti-actin (1 : 15000, Epitomics). After incubation with the primary antibodies, horseradish peroxidase-conjugated secondary antibodies were incubated to the blots at a dilution of 1 : 5000–1 : 50000 for 1 hour at room temperature. Chemiluminescence signals were detected using WesternBrightTM ECL (Advansta) and captured with Hyperfilm ECL (Amersham). Immunoblot images were quantitated using ImageJ software (NIH).

ILC2 cells were lysed by cold RIPA buffer (87787, Thermo Fisher Scientific, Waltham, MA, USA). Total protein lysates (25 μg/lane) were separated via 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were subsequently blocked with 5% nonfat milk in Tris-Buffered Saline and Tween-20 (TBST, maintaining 20 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween-20, pH 7.5) for 2 h at room temperature and further incubated overnight with rabbit anti-Hes1 (1:500, Abcam, Cambridge, MA, USA), rabbit anti-GATA3 (1:500, Abcam, Cambridge, MA, USA), rabbit anti-NF-κB (1:500), rabbit anti- Notch (1:500, Abcam, Cambridge, MA, USA), rabbit anti- RORα (1:1000, Abcam, Cambridge, MA, USA), and mouse anti-β-actin antibodies (1:800, Abcam, Cambridge, MA, USA) at 4°C according to the manufacturer’s instructions. After washing with Tris-Buffered Saline and Tween 20 (TBST, Beyotime Biotech. Shanghai, China) for three times, the membranes were incubated with the secondary antibody (Abcam, Cambridge, MA, USA) and finally processed using an enhanced chemiluminescence (ECL) reaction kit (Cell Signaling Technology, Danvers, MA, USA). The relative band densities of the target proteins relative to the β-actin band density were quantified using the Bio-Rad Quantity One 1-D analysis software package (Bio-Rad, Hercules, CA, USA).