Protease inhibitor cocktail

The right lungs of the mice were homogenized with a tissue grinder (Tiangen, Beijing, China) and then incubated in RIPA lysis buffer (Beyotime, Shanghai, China) enriched with 1 mmol/L phenylmethanesulfonyl fluoride (PMSF) (MilliporeSigma, Billerica, MA, USA) and a protease inhibitor cocktail (Servicebio, Wuhan, China) for 30 min. Following homogenization, the samples were centrifuged at 12,000× g for 15 min at 4 °C, and protein levels in the supernatants were determined using a BCA protein assay kit (Beyotime, Shanghai, China). An equal quantity of protein (20 µg) from each sample was subjected to 10% SDS-PAGE, and thereafter, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in 5% non-fat dry milk dissolved in Tris-buffered saline with 0.1% Tween-20 (TBST) to block non-specific binding sites for one hour. Following this, the membranes were incubated overnight at 4 °C with anti-phospho-ERK and total ERK primary antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA) as well as with anti-beta Actin primary antibodies (1:10,000, HUABIO, Hangzhou, China). Subsequently, the membranes underwent incubation with a secondary antibody at room temperature for 1 h. Ultimately, the blots were visualized using a chemiluminescence detection system (Advansta, Menlo Park, CA, USA).

PTC tissues as well as paired normal thyroid tissues were cut and then pulverized under liquid nitrogen. Cells were lysed with cold RIPA buffer supplemented with protease inhibitor cocktail (Servicebio), followed by ultra-sonication at low frequency and centrifugation to collect the supernatant lysate. The protein concentration was determined by Coomassie Brilliant Blue method. Following the manufacturer's protocol, the nuclei and cytoplasm proteins in PTC cells were separated using the cytoplasm and nuclear protein extraction kit (KeyGen Biotech). Twenty micrograms of proteins were separated on 10% SDS-PAGE gels and then transferred onto polyvinylidene fluoride (PVDF) membranes. Primary antibodies were diluted in 1% bovine serum albumin (BSA) and added to the PVDF membranes, which were incubated at 4℃ overnight with the following antibodies: S100A1 (Novus, NBP2-29403, 1:200), YAP (Immunoway, YT4924, 1:500), P-YAP (Immunoway, YT0708, 1:500), GAPDH (AntGene, ANT011, 1:5000), Histone H3 (Abcom, ab1791, 1:500). The PVDF membranes were then incubated with horseradish peroxidase (HRP) goat anti-rabbit (AntGene, ANT020, 1:2000) or HRP goat anti-mouse (AntGene, ANT19, 1:2000) secondary antibody. The proteins were detected using the WesternBright™ ECL kit (advansta).

GCs were washed in cold PBS and lysed with RIPA Lysis Buffer (Servicebio, Wuhan, China) containing protease inhibitor cocktail (Servicebio, Wuhan, China). Lysates were centrifuged at 12,000 × g for 15 min at 4 °C, to obtain the total cell lysates. Western Blot analysis was performed using standard protocols. Antibodies used are listed in Supplementary Table S2.

Tissues were lysed in RIPA buffer (Servicebio, Wuhan, China) containing a protease inhibitor cocktail (Servicebio). Protein samples were separated on 12% sodium dodecyl sulfate–polyacrylamide gels by electrophoresis and transferred onto nitrocellulose membranes (Servicebio). The membranes were blocked and incubated overnight at 4 °C with primary antibodies against IL-6, JAK3, p-JAK3, STAT3, p-STAT3, SOCS3 (dilution 1:1000 for all), and β-actin (dilution 1:2000). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Servicebio; 1:5000) for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Servicebio), imaged using a CLINX 6100 imaging system (ClINX, Shanghai, China), and quantified with AIWBwell™ software (Servicebio). β-actin was used as an internal control.

Briefly, approximately 50 mg of pancreatic tissues or isolated acinar cells was homogenized in RIPA buffer supplemented with a protease inhibitor cocktail (G2006, Servicebio, China) and trypsin inhibitor (XY210088, X-Y Biotechnology, China). We then used a Bicinchoninic Acid (BCA) Protein Assay Kit (#23225, Pierce) to determine the protein concentrations. Equal amounts of protein were separated on 10% Bis-Tris gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated with the primary antibodies overnight at 4 °C after blocking with 5% non-fat milk at RT for 60 min. The membranes were washed three times for 30 min with Tris-Buffered SalineTween20 (TBST), containing 0.1% Tween-20, and then incubated with secondary antibodies. After the final washes with TBST, the signals were detected using Pierce^TM Enhanced Chemiluminescence (ECL) Western Blotting Substrate (#32106, Thermo Fisher). The respective antibodies are listed in Supplementary Table S3.

Proteins of cells and lung tissues were extracted by RIPA Lysis Buffer (CoWin Biosciences, China) with phenylmethanesulfonylfluoride (PMSF; 1:100, MilliporeSigma) and protease inhibitor cocktail (1:100, Servicebio, China) and quantified by BCA protein assay kit (ECOTOP, China) for SDS-PAGE electrophoresis. Separated proteins were transferred to a polyvinylidene difluoride membrane (PVDF). The PVDF membrane was incubated with first antibodies overnight at 4 ℃ after blocked with 5% skim milk for 1 h. Antibodies against caspase-11 (ab240991, abcam, 1:1000), Gsdmd (ab219800, abcam, 1:1000), NLRP3 (15101, Cell Signaling Technology, 1:1000) and HRP-conjugated secondary antibodies (Proteintech, 1: 10,000) were used.