Gb15002

The nasal mucosa tissues from each group of mice were collected and ground to a powder. Aliquots of HNEpC and LAD2 cells that had been treated were collected. Total protein was extracted by using ice‐cold RIPA lysis buffer and the total protein concentration in each extract was determined by the BCA method. A 50 μg sample of protein from each extract was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the separated protein bands were transferred onto polyvinylidene fluoride membranes (Merck), which were subsequently blocked. Next, the membranes were incubated overnight at 4°C with primary antibodies of RORα (NBP1‐52813, 1:1000; Novus Biologicals), LC3 (PA01524, 1:1000; Boster), bECLin 1 (PB9076, 1:1000; Boster), p62 (PB0458, 1:2500; Boster), NLRP3 (27458‐1‐AP, 1:2000; Proteintech), Caspase 1 (81482‐1‐RR, 1:5000; Proteintech), ASC (10500‐1‐AP, 1:4000; Proteintech), and GAPDH (GB15002, 1:2000; Servicebio). After washing, the membranes were incubated with a secondary antibody at room temperature for 1.5 h. The immunostained protein bands were detected by enhanced chemiluminescence (ECL; Thermo). The Grayscale values of the target protein bands were analyzed using ImageJ software.

WB was conducted following the methodology described in a previous publication [47 (link)]. The following antibodies were applied: Rabbit Anti-CD9 antibody (1:1000, Abcam, UK, ab92726), Rabbit Anti-CD63 antibody (1:1000, Abcam, UK, ab134045), Rabbit Anti-CD81 antibody (1:1000, Abcam, UK, ab109201), Rabbit Anti-Calnexin antibody (1:1000, Abcam, UK, ab22595), Rabbit Anti-Caspase-9 antibody (1:1000, Abcam, UK, ab22595), Rabbit Anti-Phospho-AKT(Ser473) antibody (1:1000, Absin, abs130002), Rabbit Anti-AKT antibody (1:1000, CST, Beverly, MA, USA, 4685S), Rabbit Anti-BAX antibody (1:8000, Proteintech, 50599-2-Ig), Mouse Anti-Bcl2 antibody (1:2000, Proteintech, 68103-1-Ig), Mouse Anti-GAPDH antibody (1:1000, Servicebio, GB15002). Using Odyssey Infrared Imaging System, the original images of the membranes were recorded and analyzed according to the ECL WB Protocol (Bio-Rad, Milan, Italy).

Total IPEC-J2 cell proteins were extracted using Radioimmunoprecipitation assay (Beyotime). The extracted proteins were quantified using a bicinchoninic acid protein analysis kit (Beyotime). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) was used to separate equal amounts of proteins, which were then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies overnight at 4°C.
The primary antibodies comprised those recognizing: MX dynamin like GTPase 1 (MX1) (bsm-51528m, Bioss, Woburn, MA, USA; 1:1,000); Janus kinase 1 (JAK1) (bs-1439R, Bioss; 1:1,000); phosphorylated (p-JAK1) (bs-3238R, Bioss; 1:1,000); signal transducer and activator of transcription 3 (STAT3) (GB11176, Servicebio, Wuhan, China; 1:1,000); p-STAT3 (bs-1658R, Bioss; 1:1,000); and GAPDH (GB15002, Servicebio; 1:2,000). After incubation with horseradish peroxidase-labeled secondary antibodies (GB23303, Servicebio; 1:5,000) or (GB25301, Servicebio; 1:5,000), the immunoreactive protein bands were observed using the enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).

Kidney tissues and HK-2 cells were placed on ice and lysed by adding RIPA lysis solution, PMSF and phosphatase inhibitor (all from Servicebio, Wuhan, China) for 30 min. After the protein concentration was determined by BCA method (Beyotime Biotechnology, Shanghai, China), 20 μg of protein was taken for SDS-PAGE electrophoresis, electrotransferred to PVDF membrane, put into TBST containing 0.1% Tween-20 and 5% skim milk for 1 h. The primary antibodies against these targets, YAP (14074, CST, Danvers, MA, USA), ACSL4 (ET7111-43, Huabio, Hangzhou, China), Collagen Ⅰ (ab34710, Abcam, Cambridge, UK), fibronectin (ab2413, Abcam, Cambridge, UK), α-SMA (GB111364, ServiceBio, Wuhan, China), P53 (ab26, Abcam, Cambridge, UK), GPX4 (ET1706-45, Huabio, Hangzhou, China), SLC7A11 (HA600098, Huabio, Hangzhou, China), GAPDH (GB15002, ServiceBio, Wuhan, China), Histone H3 (GB11102, ServiceBio, Wuhan, China), and incubated at 4 °C overnight. The secondary antibodies containing goat anti-mouse/rabbit IgG polymer (SA00001-1, SA0001-0, proteinTech, Wuhan, China) were added and incubated at 37 °C for 1 h. After washing with TBST, Chemi Doc imaging system (Bio-Rad, Hercules, CA, USA) was used to detect protein blots, and Image J software, version 1.52a (NIH, Bethesda, USA) for semi-quantitative analysis.

Total protein was obtained by lysing HBE cells with RIPA buffer containing 1% PMSF and the protease inhibitor cocktail, and total protein was determined by the BCA method. The samples were transferred to PVDF membranes. The PVDF membranes were blocked for 2 h with 10% skim milk. The primary antibodies were incubated overnight at 4°C, and the primary antibodies used as follows, GAPDH (1:1000, GB15002, Servicebio, Wuhan, China), β-actin (1:1000, GB15003, Servicebio), NF-kB (1:1000, GB11997, Servicebio),PTGS2 (1:2000,27308-1-AP, Proteintech, Wuhan, China), LaminB (1:2000, GB111802, Servicebio), and Ubiquitin (1: 500, AG3164, Beyotime, Shanghai, China). The membrane was then incubated for 2 h with secondary antibodies. Immunoreactive bands were displayed and visualized with an image capture system (ChemiDoc MP, Bio-Rad).