Sc 374639

RIPA buffer, phosphatase, and protease inhibitors (MEC, China) was added to mouse lung or cell samples, and being lysed on ice plate. An equal amount of protein sample was added to each well for electrophoresis in SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane (Millipore, USA). At room temperature, the membrane was washed with TBST and sealed in 5% skimmed milk for 60 min. The membrane was washed three times for 5 min each time and then incubated with primary antibodies against RIP1 (1:500 dilution, 3493, Cell Signaling Technology, USA), RIP3 (phospho S232) (1:500 dilution, ab195117, Abcam, USA), RIP3 (1:500 dilution, sc-374639, Santa-Cruz, USA), MLKL (phospho S345) (1:1,000 dilution, ab196436, Abcam, USA), MLKL (1:500 dilution, sc-293201, Santa-Cruz, USA), and GAPDH (1:1,000–1:2,000 dilution, CST, USA) at 4°C overnight. After membranes were incubated with appropriate secondary antibodies for 1 h at room temperature, protein bands were visualized using enhanced chemiluminescence (Biosharp, Shanghai, China). Signal quantification was performed with ImageJ software.

The stented esophagus was harvested from rats euthanized either 15 or 28 days after SEMS placement. The tissue samples, once fixed and embedded in paraffin, were sectioned into 4 μm slices and stained with hematoxylin and eosin for histological analysis. For immunohistochemical analysis, sections from animals euthanized at 15 days after SEMS placement were deparaffinized, rehydrated, and incubated with a primary antibody against heat shock protein 70 (HSP70) (ab2747; Abcam, Cambridge, UK; diluted 1:400). Conversely, sections from 28-day euthanized animals underwent similar IHC preparation and were stained using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (ApopTag; Qbiogene, Darmstadt, Germany) and a primary antibody against Receptor-interacting kinase 3 (RIP3) (sc-374639; Santa Cruz Biotechnology, CA, USA; diluted 1:50). After staining, sections were examined using a digital slide scanner (Aperio ScanScope CS; Leica Biosystems, CA, USA). Epithelial and submucosal layer thickness and percentages of TUNEL-, HSP70-, and RIP3-positive cells were assessed using ImageJ v1.53 (NIH, MD, USA). Inflammatory cell infiltration was graded from 1 (mild) to 5 (severe). Data were averaged from 2 segments (proximal and distal) and 6 points on each segment’s circumference. Two blinded observers reached consensus on results.

The cells were collected as previously described in the western blotting section. Cells were lysed in binding buffer (50 mM Tris–Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitor). The monoclonal antibody against RIPK1(CA#sc-133102, mouse, Santa Cruz, CA, United States) or against RIPK3 (CA# sc-374639, mouse, Santa Cruz, CA, United States) or a matched-isotype antibody IgG (CST) was incubated with Protein A/G Magnetic Beads (MCE, Monmouth Junction, NJ, United States) at 4°C overnight. On the second day, a total of 1 mg protein sample lysate was incubated with an antibody-beads complex for more than 8 h. Then, the magnetic rack was used to precipitated the antibody-beads complexes. Then, the complexes were washed for three times and then eluted from beads by boiling at 95°C with 4 × LDS loading buffer (CA#161-0747, Biorad, United States). Finally, these protein samples were resolved by SDS/PAGE assay and immunoblotted with antibodies as indicated.

The lung homogenates and cells were lysed in RIPA buffer (Beyotime) containing protease inhibitor (Bimake). Total protein was measured with Pierce BCA assay and equal amounts of denatured proteins were loaded and run on 10 to 12.5% gradient gel. After transferred to PVDF membranes, the protein was hybridized with indicated primary antibodies and corresponding HRP labeled secondary antibodies. Super ECL Substrate (Tanon, Shanghai, China) was used to detect the bands, which were analyzed and quantitated using Image J software. The primary antibody used in western blot analysis were as follows: Anti-tubulin (11224-1-AP; proteintech); anti-GAPDH (10494-1-AP; proteintech); anti-PTRF (ab48824; Abcam); anti-ZBP1 (sc-271438; Santa Cruz Biotechnology); anti-RIP3 (phospho T231 + S232) (2D7, ab205421; Abcam); anti-RIP3 (sc-374639; Santa Cruz Biotechnology); anti-mouse MLKL (phospho S345) (EPR9515(2), ab196436; Abcam); anti-human MLKL (phospho S358) (EPR9514, ab1187091; Abcam); anti-MLKL (ab243142; Abcam); anti-IL-1β (ab9722; Abcam); anti-caspase-3 (9662 S; CST); anti-GSDMD (ab209845; Abcam); anti-mouse IL-33 (AF3626; R&D Systems); anti-human IL-33 (ab54385; Abcam). The original Western blotting gel images are listed in the Supplementary Materials.