Odyssey infrared scanning device

NECs were homogenized and lysed in RIPA lysis buffer (Sigma-Aldrich; Merck KGaA) with protease and phosphatase inhibitors. Protein concentration was determined using BCA reagent (Beyotime Institute of Biotechnology). Protein samples (40 µg) were separated by SDS-PAGE on 12% gel, and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were then blocked in 5% low-fat milk for 1 h at room temperature, following which the membranes were incubated with primary antibodies at 4°C overnight, followed by incubation with a goat anti-rabbit secondary antibody (1:10,000; cat. no. 926-32211; LI-COR Biosciences) at room temperature for 2 h. The membranes were washed with TBS with 0.1% Tween-20 and scanned using an Odyssey infrared scanning device (LI-COR Biosciences). The Odyssey^® CLx Imaging System (LI-COR Biosciences) was used to detect the expression level of each protein. Rabbit anti-mouse TSLP (1:1,000), STAT6 (1:1,000), p-STAT6 (1:1,000) and GAPDH (1:1,000) antibodies were purchased from Cell Signaling Technology, Inc. Rabbit anti-mouse IL-13Rα1 (1:1,000) antibodies was purchased from Affinity Biosciences. Band intensities (gray values) were measured with ImageJ 1.52 (National Institutes of Health), and GAPDH was used as the internal reference.