Caspase 1 p20

Oesophageal strips or cultured cells were collected to extract the protein with RIPA lysis buffer (Beyotime Biotech), which contained 1 mmol/L final concentration of phenylmethanesulfonyl fluoride (PMSF). After complete lysis, the samples were centrifuged at 10 000 g for 5 minutes at 4°C to precipitate the tissue debris. The supernatants were used to measure the protein concentration by a BCA Protein Assay Kit (Beyotime Biotech). The proteins were electrophoresed in 10% SDS‐PAGE gels and then transferred to PVDF membranes. After blocking with 5% skim milk for 1 hour at room temperature, the membranes were incubated with the following primary antibodies: CaSR (1:200, ab19347, Abcam), NLRP3 (1:100, ab214185, Abcam), ASC (1:100, sc‐22514‐R, Santa Cruz), Caspase‐1 p20 (1:100, sc‐398715, Santa Cruz) and IL‐1β (1:100, sc‐32294, Santa Cruz) at 4°C overnight. The membrane was washed with TBST and incubated with secondary antibodies for 2 hours at room temperature. Protein bands were visualized on the membrane with a Gel Imaging System, and the protein bands were quantified with ImageJ software.

Part of the right lobe of the mouse liver tissue was weighed and cut into pieces. The mouse liver tissue sample or AML12 cells were lysed on ice in RIPA buffer (Roche Diagnostics, Basel, Switzerland) containing a mixture of phosphatase inhibitor and serine protease inhibitor for 30 min. After that, the lysate was centrifuged at 12,000 g for 15 min at 4 °C. The sediment was discarded, and the supernatant was taken. The BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentration in the supernatant. The protein was denatured and separated by a 10% SDS-PAGE gel, and then transferred to a PVDF membrane (Miripoli, Massachusetts, USA). At room temperature, the membrane was sealed with 5% (w/v) skimmed milk for 2 h. Next, the membrane was incubated with the primary antibody overnight at 4 °C: ERK, p-ERK, HO-1, GPX4, NLRP3, Gasdermin-D (Cell Signaling Technology, MA, USA), Caspase 1 (p20) (Santa Cruz, CA, USA), which would combine HRP. The membrane was incubated with the secondary antibody for 2 h, the membrane was washed five times with TBST, and the protein was visualized using an enhanced chemiluminescence method (Yeasen, Shanghai, China). Imager (Tanon, Shanghai, China) was used to image and quantify the blot.