Nonfat milk

Total protein were collected from AGS and NCI-N87 cells in a radioimmunoprecipitation assay buffer (Beijing Solarbio Science and Technology Co., Ltd.). The protein concentration was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 30 µg protein was separated on 10% SDS-PAGE gels, followed by transfer of electrophoresed proteins onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk (Beyotime Biotechnology, Beijing, China) and incubated with primary antibodies, including GPX4 (no. 52455, 1:1,000, Cell Signaling Technology, Inc.), xCT (no. 12691, 1:1,000, Cell Signaling Technology, Inc.), NCOA4 (no. 66849; 1:1,000; Abcam), FTH-1 (no. 4393; 1:1,000; Abcam), and GAPDH (cat. no. 5174, 1:4,000, Cell Signaling Technology, Inc.), at 4°C overnight. Subsequently, the cells were incubated with the corresponding peroxidase-conjugated secondary antibodies (both 1:5,000; cat. no. ZB-2301 and ZB-2305; Beijing Zhongshan Golden Bridge Biotechnology Co.). The protein bands were detected using enhanced chemiluminescence reagent (EMD Millipore). Relative protein expression was normalized to GAPDH. All the experiments were repeated three times. ImageJ 1.43b software (National Institutes of Health) was used for the densitometry analysis.

RIPA buffer was used to lyse mouse ear tissue samples as above, after which protein levels therein were measured via BCA assay. Equal protein amounts were then separated via 6-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific, MA, USA). These blots were then blocked for 2 hours using 5% non-fat milk (Beyotime, Shanghai, China) in 0.1% Tween 20 in tris-buffered saline (TBS-T) at 26 °C. They were then incubated overnight at 4 °C while being constantly shaken with primary antibodies specific to β-actin (HRP-60008, Proteintech, IL, USA), TLR3 (ab62566, Abcam, Cambridge, MA, USA), IL-13Rα1 (ab79277, Abcam, Cambridge, MA, USA), or TSLP (ab115700, Abcam, Cambridge, MA, USA). Blots were then washed repeatedly, probed for 2 hours with secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (ab205718, Abcam, Cambridge, MA, USA), with β-actin being assessed as a loading control. Protein bands were then detected using enhanced chemiluminescence (ECL) detection reagents (Thermo Fisher Scientific, MA, USA) with a Tanon 5200 automatic chemiluminescence image analysis system (Tanon, Shanghai, China).

The total cellular proteins were extracted with RIPA buffer (Beyotime). Equal amounts of protein (30 μg/lane) were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA). After blocking with PBS buffer (Beyotime) containing 5% non-fat milk and 0.1% Tween 20 (Beyotime), membranes were incubated with primary antibody over-night at 4℃. Subsequently HRP-conjugated secondary antibodies were incubated for 1 hour at room temperature and developed with enhanced chemiluminesence (Beyotime). All antibodies used were listed in S1 Table.

For extraction of protein, RIPA lysis buffer (Keygen) was used to lyse cells or tissues. After measuring the protein concentration using a BCA protein assay kit (Abcam), electrophoresis was performed with SDS‐PAGE to separate the protein. Next, the protein was transferred onto PVDF membranes (Invitrogen), followed by blocking in nonfat milk (5%; Beyotime). After that, the membranes were probed with primary antibodies (overnight, 4°C). The primary antibodies containing Cyclin D1 (1:1000, ab226977), MMP9 (1:2000, ab76003), PDL1 (1:1000, ab205921) and GAPDH (1:2000, ab9485) were all provided by Abcam. After incubation with secondary antibody (1:3000, ab205718, Abcam), enhanced chemiluminescence reagent (Abcam) was used to visualize the blot signal.