Cleaved il 1β

Antibodies against IL-1β and cleaved IL-1β were purchased from Cell Signaling Technology (Danvers, MA, USA). Caspase-1 and cleaved Caspase-1 (p20) were purchased from Abcam (Cambridge, UK).

Western blotting was performed as described previously (Mohammed et al., 2021 (link)). Images were taken using a Chemidoc imager (Bio‐Rad) and quantified using ImageJ software (U.S. National Institutes of Health). Primary antibodies against the following proteins were used: phospho(S345) MLKL, phospho(T231+ S232)‐RIPK3, HMGB1 and TNFα from Abcam; RIPK1 and RIPK3 from Novus Biologicals; MLKL from Millipore Sigma; Phospho (Ser166)‐RIPK1, Cleaved Caspase‐3, Caspase 3 and Cleaved IL‐1β from Cell Signaling Technology (Danvers, MA); desmin from ThermoFisher Scientific; GAPDH, β‐tubulin and β‐actin were from Sigma‐Aldrich. HRP‐linked anti‐rabbit IgG, HRP‐linked anti‐mouse IgG and HRP‐linked anti‐rat IgG from Cell Signaling Technology (Danvers, MA) were used as secondary antibodies.

Exosomes or cells were lysed with RIPA buffer containing a complete protease inhibitor tablet (Roche). Proteins were separated by SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes. After blocked in 5% skim milk for 30 min, membranes were probed with various primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase–linked secondary antibodies for 1 h at room temperature, and visualized with electrochemiluminescence by the chemiluminescence instrument. The following antibodies were used: TRIM59 (Invitrogen, Cat: PA5–38726), ABHD5 (Invitrogen, Cat: PA5–78704), CD63 (Invitrogen, Cat: 10628D), CD81 (Invitrogen, Cat: 16–0811-82), TSG101 (Invitrogen, Cat: PA5–82236), SLC16A4(MCT4) (Invitrogen, Cat: PA5–80008), β-actin (Cell Signaling Technology, Cat: #3700), HA-Tag (Cell Signaling Technology, Cat: #3724), Myc-Tag (Cell Signaling Technology, Cat: #2276), IL-1β (Cell Signaling Technology, Cat:#12703), Cleaved-IL-1β (Cell Signaling Technology, Cat: #83186), Caspase-1(Cell Signaling Technology, Cat: #3866), and Cleaved Caspase-1 (Cell Signaling Technology, Cat: #89332).

The cultured cells or skin tissues were lysed in RIPA lysis buffer with protease and phosphatase inhibitors. The protein concentration was quantified by bicinchoninic acid (BCA) assay (Boster, California, United States). The protein samples were resolved by 10% SDS-PAGE gels and then transferred to PVDF membranes (Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland). After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight. The membranes were then incubated with HRP-conjugated anti-rabbit/mouse IgG (111-035-003/115-035-003, Jackson ImmunoResearch, West Grove, United States) secondary antibodies for 1 h at room temperature. Images were visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology).

Protein concentration was determined by the BCA protein assay [43 (link)]. Membranes were exposed to the following antibodies purchased from Cell Signaling Technology: Occludin (#91131), Claudin-2 (#48120), Cleaved-IL-1β (#63124), IL-18 (#57058), Caspase-1 (#2225), NLRP3 (#15101), ASC/TMS1 (#67824), AIM2(#63660), β-actin (#4970), Malt1(#2494), Bcl10(#4237), Nod1(#3545), Card9 (#12283), Ripk2 (#4142). Anti-ZO-1 antibody purchased from abcam (ab221547), Anti-NLRC4 antibody purchased from ECM Bioscience(#NP5381). Western blotting signals were quantified by a FluorChem densitometer (Alpha Innotech, San Leandro, CA).

Cells used for Western blotting were stimulated in FBS-free media. Cells were lysed in RIPA lysis buffer (Merck Millipore, Burlington, MA), and the protein concentrations were measured using the Micro BCA™ Protein Assay (Thermo Scientific). The proteins were separated in 8-16% stain-free TGX gels (Bio-Rad, Hercules, CA), transferred to PVDF membranes (iBlot® 2 PVDF regular stacks, Invitrogen, Carlsbad, CA), and analysed by immunoblotting. The primary antibodies used were cleaved IL-1β (1 : 1000 Cell Signaling Technologies, Danvers, MA. cat.nr:12242), IL-18 (1 : 1000 Abcam, Cambridge, UK. cat.nr: EPR19954-188), and caspase-1 p20 (1 : 750 Adipogen Life Sciences, cat.nr:AG-20B-0048-C100). Secondary antibodies used were goat anti mouse (1 : 5000 Abcam cat.nr: Ab6789), rabbit anti goat (1 : 2000 Dako, Agilent. Santa Clara, CA. cat.nr: P0160), and goat anti-rabbit (1 : 3000 Invitrogen cat.nr: A11034). Membranes were washed in TBST buffer and analysed with ChemiDoc™ MP Imaging System (Bio-Rad).

Immunofluorescent staining was performed according to the protocol of 'Immunofluorescent Staining of Paraffin-Embedded Tissue (Novus Biologicals)'. The information of antibodies is shown as follows: cleaved IL-1β (Cell Signaling), Caspase-3 (Santa Cruz), Phospho-p70s6k (Cell Signaling), Phospho-EIF2α (Cell Signaling), EIF3A (Cell Signaling), vWF (Abcam), VCAM-1 (Cell Signaling) and p22^phox (Cell Signaling).