Ab234437

The protein suspension of liver tissues for Western blotting was obtained using a total protein extraction kit (APPLYGEN P1250, Beijing, China) and protein concentrations were determined by BCA protein assay kit following the manufacturer’s instructions. CD68 (ab283654, Abcam), HSP90 (ab59459, Abcam), HSF1 (ab242138, Abcam), SGT1 (11019-2, Proteintech), IL-1β (ab234437, Abcam), Caspase 1 (ab179515, Abcam), cleaved-IL-1β (#63124, CST), cleaved-caspase-1 (#89332, CST), NLRP3 (ab263899, Abcam), ASC (#67824, CST), GAPDH (#5174, CST), and β-actin (sc-69879, Santa Cruze) anti-bodies were used and blots were quantified using Image J Software (NIH, Bethesda, MD, USA). Each group was tested using three samples that were created by mixing the liver samples from mice numbered 1–3, 4–6, and 7–8, respectively. Western blot results were normalized to the GAPDH band or the β-actin band.

Protein lysates of cell samples or NP tissues were prepared by RIPA lysis buffer plus 1% phenylmethanesulfonyl fluoride. The total protein concentrations were measured with a BCA protein assay kit (Santa Cruz). Proteins were then separated by 10% SDS‐PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were then blocked by 5% bovine serum albumin for 2 hours at room temperature, followed by incubation with primary antibodies at 4°C overnight. After that, the membranes were incubated with second antibodies (1:5000, Abcam, ab150077) at room temperature for 2 hours. Protein bands were then detected using an enhanced chemiluminescence kit (ThermoFisher) and quantified by Image J software. Antibodies against NLRP3 (1:2000, ab214185), IL‐18 (1:1000,ab71495), IL‐1β (1:2000, ab234437), Gasdermin D (GSDMD, 1:1000, ab219800), TSG101 (1:2000, ab125011), and GAPDH (1:5000, ab181602) were purchased from Abcam. Antibodies against CD9 (1:2000, #13403), CD81 (1:2000, #56039), CD63 (1:2000, #55051), GM130 (1:1000, #12480), Cleaved caspase‐1 (1:1000, #89332), cleaved GSDMD (1:1000, #50928) and Calnexin (1:1000, #2679) were purchased from CST.

Melatonin was purchased from Nacalai Tesque (Kyoto, Japan). Murine macrophage cell line, RAW264.7, was cultured in RPMI1640 supplemented with 5% FBS. Transfecting (PH)-PLCδ-GFP plasmid (kindly gifted by Dr. Greg Fairn, Dalhausie Univeristy, Canada) into cells was performed with Neon Transfection System (Thermo Fisher Scientific, Massachusetts, USA). For the Neon transfection, RAW 264.7 cells were transfected with the mixture of plasmid DNA and resuspension buffer and then electroporated with the setting of 1680 V, 20 ms, 1 pulse. Cells were transferred to glass coverslip with RPMI1640 supplemented with 10% FBS for 2 days before microscope observation. For optimal mitochondrial condition, cells were used for all experiments after 3 days of cellular passage. For the hypoxia experiment, RAW264.7 cells were pretreated in an anaerobic chamber for 6 h at 36.5 °C (O₂ < 0.1%). The cells were then treated with Melatonin (1 mM) and infected by EMCV for 16 h. Polyinosinic-polycytidylic acid (poly (I:C), LMW) was purchased from InvivoGen (California, USA). Lipofectamine 3000 (Thermo Fisher Scientific) was used for lipofection for poly (I:C). For immunoblotting, anti-mouse IL-1β antibody (rabbit monoclonal, ab234437, Abcam, Cambridge, UK) and anti-pan actin antibody (mouse monoclonal, ACTN05(C4), ab3280, Abcam) were used.

The vaginal tissues were lysed and homogenized in RIPA buffer with phenylmethylsulfonyl fluoride (PMSF) and quantified with a BCA kit (Keygen Biotech, KGP902, China). The protein samples (30 μg/lane) were separated using SDS-PAGE (Beyotim, P0012A, China) and transferred to polyvinylidene difluoride membranes (PVDF) (Merck, IPVH00010, United States) after electrophoresis. The membranes were blocked with 5% skimmed milk for 3 h, followed by incubation with TNF-α (1:1,000, Abcam, ab205587, United States), IL-1β (1:1,000, Abcam, ab234437, United States), IL-6 (1:1,000, Abcam, ab229381, United States), Dectin-1 (1:1,000, Abcam, ab140039, United States), Syk (1:1,000, Cell Signaling Technology, 13198T, United States), PLCγ-2 (1:1,000, Cell Signaling Technology, 3872T, United States), CARD9 (1:500, Affinity, DF8387, China), NF-κB (1:1,000, Cell Signaling Technology, 8242T, USA) and β-actin (1:2,000, Affinity, AF7018, China) antibodies overnight at 4°C. Then, the membranes were incubated with an HRP-conjugated antibody (1:5,000, Affinity, S0001, China) at room temperature for 1 h. The membranes were visualized with a chemiluminescence (ECL) kit by a Tanon 5200 system (Shanghai, China). ImageJ software (Version 1.52a) was used to measure the protein bands based on that of β-actin.

Proteins were isolated from the frozen cardiac tissue, and their concentration was calculated using the bicinchoninic acid (BCA) method (Thermo Fisher Scientific, Waltham, MA, USA; A53225). Immunoblotting was performed as previously described [15 (link)]. In this study, the primary antibodies used recognized NLRP3(1 : 1000, CST, 15101S), Cle-caspase-1 (1 : 1000, Abcam, ab1789515), ASC (1 : 1000, CST, 67824), thioredoxin-interacting protein (TXNIP) (1 : 1000, CST, 14715), caspase-3 (1 : 1000, CST, 9662), BCL-2 apoptosis regulator (Bcl-2) (1 : 1000, CST, 3498), IL-1β (1 : 1000, Abcam, ab234437), Bax(1 : 1000, Abcam, ab32503), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000, Proteintech, 60004-1-Ig). Protein bands were visualized using the chemiluminescence system (Tanon Science & Technology Co., Shanghai, China) for the required time. Quantitative analysis was performed using Image J software (NIH, Bethesda, MD, USA).

The carotid arteries specimens were embedded in the OCT compound and serially sectioned (5 μm). Hematoxylin and eosin (H&E), Masson's trichrome, and Sirius red staining were performed according to the manufacturers' protocols (Sigma). To evaluate the lipid content in the vasculature, sections were stained with oil red O, using Mayer's hematoxylin (Sigma) as the counter stain.
For immunofluorescence analysis, serial paraffin sections were incubated with different primary antibodies overnight at 4°C, followed by the corresponding fluorochrome-conjugated antibodies for detection. The following primary antibodies were used: rat monoclonal antibody against CD68 (Abcam, ab53444), rabbit monoclonal antibody against NLRP3 (Abcam, ab270449), rabbit monoclonal antibody against IL-1β (Abcam, ab234437), rabbit polyclonal antibody against caspase-1 (ABclonal, A16792), and rabbit monoclonal antibody against NR1D1 (Abcam, ab174309). After they reacted with the corresponding Alexa Fluor 488 or 594-conjugated secondary antibodies (Invitrogen), DAPI was added to visualize nuclei. Fluorescent images were acquired with a laser scanning confocal microscope (Leica TCS SP8, Leica Microsystems). All the images and staining intensities were acquired and measured with Image-Pro Plus 6.0 (Media Cybernetics Inc.).

Proteins in cell culture media and hippocampal tissues were lysed in RIPA lysis buffer with protease inhibitor cocktail. Protein samples were subjected to 10% SDS‐PAGE and transferred to PVDF membranes. The membranes were subsequently incubated with antibodies iNOS (Abcam #ab178945, USA, 1:500), IL‐1β (Abcam #ab234437, 1:500), IL‐6 (Abcam #ab233706, 1:500), TNF‐α (Abcam #ab215188, USA, 1:500), c‐Jun (CST #9165, USA, 1:500), p65(CST #8242, USA, 1:500), IRAK1 (CST #4504, USA, 1:500), β‐actin (CST #3700, USA, 1:2000). Then the membranes were incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature and detected with enhanced chemiluminescence. The results were analyzed by using ImageJ software (National Institutes of Health, USA).​ The original western blot images have been attached in the Appendix S1.