Bicinchoninic acid kit

A small piece of ear tissue was lysed with RIPA Lysis Buffer (Solarbio, Beijing, China) using Qiagen TissueLyser II (Qiagen, HK, China) with shaking at 20 Hz for 5 min and standing on ice for 5 min. The aforementioned procedure was repeated twice. Then, the lysates were centrifuged at 13,000 ×g for 10 min. Concentrations of total protein were determined using a bicinchoninic acid kit (Solarbio). Subsequently, an equal amount of protein sample was loaded in each well and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for electrophoresis and then transferred onto polyvinylidene difluoride membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with the following primary antibodies: mouse anti-TSLP (1:1000, Abcam), mouse anti-nuclear factor-kappa B (NF-κB) p65 (1:1000, Cell Signaling Technology, MA, USA), and mouse anti-PAR2 (1:1000, Abcam); anti-β-actin antibody was used as a loading control. The other day, the membrane was incubated with corresponding secondary antibodies after washing. Finally, specific bands were observed by ImageQuant 350 (GE, MA, USA) with standard chemiluminescence (Thermo, IL, USA). The band intensity was calculated by ImageJ software (National Institutes of Health, MD, USA) compared with β-actin.

Proteins were extracted with RIPA lysis buffer (CWBiotech, Beijing, China) on ice for 30 minutes. Protein quantity was determined using bicinchoninic acid kit purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Equivalent amounts of proteins (30 µg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 hour at room temperature and then incubated with the following specified antibodies for 1 hour at room temperature: anti-MTBP (1:2,000 dilution; Sigma-Aldrich), anti-ZEB2 (1:5,000 dilution; Abcam), anti-E-cadherin (1:5,000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (1:5,000 dilution; Cell Signaling Technology), anti-β-catenin (1:5,000 dilution; Cell Signaling Technology), anti-Vimentin (1:5,000 dilution; Cell Signaling Technology), and anti-GAPDH (1:5,000 dilution; Cell Signaling Technology). The membranes were then incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG (ZSGB-BIO) for 1 hour at room temperature. Signals were visualized using an enhanced chemiluminescence reagent and captured using AI600 version 1.2.0 on Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).