Ag 20b 0014

LPS (L2630) and ATP (A7699) were purchased from Sigma-Aldrich. Nigericin (HY-127019), JANEX-1 (HY-15508), and A419259 (HY-15764) were from MedChemExpress (Monmouth, NJ, USA). Vigofect (T001) was from Vigorous (Beijing, China). The Lipofectamine™ RNAiMAX transfection reagent was from ThermoFisher (Waltham, MA, USA). The anti-rabbit ASC antibody (1:1000; #67824) was from Cell Signaling Technology (Cambridge, MA, USA). The anti-mouse NLRP3 (1:1,000; AG-20B-0014) and anti-mouse caspase 1 (p20) (1:1,000; AG-20B-0042) antibodies were from AdipoGen (San Diego, CA, USA). The anti-mouse IL-1β antibody (1:1,000; AF-401-NA) was from R&D Systems (Minneapolis, MN, USA). The anti-rabbit HCK antibody (1:500; A14537) was from Abclonal (Wuhan, China) and anti-rabbit HCK antibody (1:200; 11600-1-AP) was from ProteinTech (Wuhan, China). Anti-mouse β-tubulin (1:2,000; CW0098A), anti-mouse GAPDH (1:3000; CW0100M), goat-anti mouse horse radish peroxidase (HRP) IgG(H + L) (1:5,000; CW0110S), and goat anti-rabbit HRP IgG (H + L) (1:5,000; CW0103S) were from CWBiotech (Beijing, China). The anti-mouse FLAG antibody (1:1,000; F1804) was from Sigma-Aldrich and anti-mouse MYC antibody (M047-3) was from MBL (Woburn, MA, USA). Goat anti-mouse IgG HRP (L) and goat anti-mouse HRP(Fc) were from Invitrogen.

Tanshinone I (Tan I, HY-N0134), MCC950 (HY-12815 A), and nigericin (HY-127019) were from MedChemExpress. ATP, SiO₂, Pam3CSK4, poly (I:C), poly (dA:dT), and ultrapure lipopolysaccharide (LPS) were from Invivogen. Phorbol 12-myristate 13-acetate (PMA, P8139) and DMSO (D2650) were from Sigma Aldrich. The Starfect high-efficiency transfection reagent (C101-10) was from GenStar. Antibodies were used as follows: anti-mouse IL-1β (1:1000, AF-401-NA, R&D), anti-mouse caspase-1 (1:1000, AG-20B-0042, Adipogen), anti-human cleaved IL-1β (1:2000, 12242, Cell Signaling Technology), anti-human caspase-1 (1:2000, 4199 S, Cell Signaling Technology), anti-NLRP3 (1:2000, AG-20B-0014, Adipogen), anti-ASC (1:1000, sc-22514-R, Santa Cruz), anti-Flag (1:2000, 20543-1-AP, Proteintech), and anti-GAPDH (1:5000, 60004-1-Ig, Proteintech).

At 24 h after tMCAO, mice were anesthetized with ketamine/xylazine and transcardially perfused with 30 mL of PBS followed by 50 mL of 10% formalin (Fischer Scientific). Brains were removed and post-fixed in the same fixative overnight at 4 °C and then with 30% sucrose in PBS for 72 h. The brains were sectioned in the coronal plane at a thickness of 10 μm. Sections were blocked with Serum-Free Protein Block (X0909, DAKO) followed by incubation with primary antibodies against NLRP3, cleaved caspase-1 (1:200; AG-20B-0014, 1: 250; AG-20B-0042, Adipogen life sciences) and cleaved IL-1β (1:100; CST-12242, Cell signaling technology, USA) overnight at 4 °C in a humid chamber. After washing, slides were incubated with fluorescent anti mouse secondary antibodies (1:200; 072–04–18–03; Dylight-549, KPL) for 1 h at room temperature and mounted with ProLong™ Diamond Antifade Mountant with DAPI (Invirogen), and viewed using a Zeiss 710 confocal laser scanning microscope. Negative controls were prepared by omitting the primary antibodies.

For WB analysis, we used peri-infarct (penumbra) cortical regions. Using a brain matrix, the brains were rapidly dissected into 4.0 mm coronal sections (approximately 0.5 mm and −3.5 mm from bregma). Brain tissue was homogenized and processed for western blotting as previously described. Fifty-microgram of proteins were loaded in each lane and separated followed by transfer to nitrocellulose membranes. The membranes were blocked for non-specific binding and probed with primary antibodies against NLRP3, Caspase-1, ASC (1:1000; AG-20B-0014; AG-20B-0042; AG-25B-0006 Adipogen life sciences), cleaved IL-1β, Caspase-3, Phospho- NFκBp65, NFκBp65 (1:1000; CST-12242; 9664; 3033; 8242; Cell signaling technology), Cleaved PARP, Anti IL-18, IL-1β (1:1000; ab32064; ab71495; ab9722; Abcam, USA), phospho IκB (1:1000; Santa cruz biotechnology, SC8404) at 4 °C for overnight. Following TBS-T washes, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (1: 10,000; Sigma). The bands were then visualized by means of an enhanced chemiluminescent substrate system (Thermo fisher scientific). Protein levels were analyzed densitometrically, using Image J software and were normalized to loading controls, and expressed as fold change.

PMs were seeded in 6-well plates at a density of 3 × 10⁶ cells/well, and pre-treated for 1 h with NAC, then infected with N. caninum (MOI of 3:1 2:1 or 1:1). In some experiments, PMs were incubated with N. caninum for 8 h, then treated with PG at different concentrations. At 24 or 36 h post-infection, the cell lysates and supernatants were collected and assessed by western blotting, as described previously [27 (link)]. The following primary antibodies were incubated overnight at 4 °C: anti-mouse IL-1β (p17; 1/1,000; AF-401; R&D, Minneapolis, MN, USA); anti-mouse caspase-1 (p20; 1/1,000; AG-20B-0042; Adipogen, Liestal, Switzerland); anti-NLRP3 (1/1,000; AG-20B-0014, Adipogen); and anti-mouse β-actin (1/2,000; 60008–1; Proteintech, Wuhan, China). Secondary horseradish peroxidase (HRP)-conjugated rabbit anti-goat or anti-mouse IgG (1/5,000; Proteintech) was then added for 1 h at room temperature (RT). Finally, proteins were visualized using an Enhanced chemiluminescence (ECL) Western Blot Detection System (Clinx Science Instruments, Co., Ltd., Shanghai, China).

Samples for immunoblotting of caspase-1 were prepared by mixing the cell lysates with culture supernatants (lysis buffer: 5% NP-40 solution in water supplemented with 10 mM DTT and protease inhibitor solution at 1× final concentration); samples for all other protein immunoblotting were prepared without the supernatants in RIPA lysis buffer. Samples were mixed and denatured in loading buffer containing SDS and 100 mM DTT and boiled for 12 min. SDS-PAGE–separated proteins were transferred to PVDF membranes and immunoblotted with primary antibodies against IL-1α (503207, Biolegend), IL-1β (12426, Cell Signaling Technology), caspase-1 (AG-20B-0042, Adipogen), NLRP3 (AG-20B-0014, Adipogen), GSDMD (ab209845, Abcam), and β-Actin (sc-47778 HRP, Santa Cruz), Appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (anti-Armenian hamster [127-035-099], anti-mouse [315-035-047], and anti-rabbit [111-035-047], Jackson ImmunoResearch Laboratories) were used as described previously (56 (link)). Immunoblot images were acquired on an Amersham Imager using Immobilon^® Forte Western HRP Substrate (WBLUF0500, Millipore).

To analyse the expression of NLRP3 inflammasome, primary PMs were incubated for 3 h after seeding in the culture plate. Macrophages were assayed in opti‐MEM media (Gibco, 31985) in the presence or the absence of trehalose or saccharin 15 min prior to LPS stimulation (Invivogen, tlrl‐eklps; 10 ng/ml) for 3 h followed by adding ATP (Sigma–Aldrich, A6419; 5 mM) for 30 min to release IL‐1β. To confirm Atg7 protein deficiency in macrophage of Atg7^fl/fl or Atg7^fl/fl;Lyz2‐Cre mice, PMs were harvested, seeded, incubated for 3 h, and the adherent macrophages were collected for further analysis. Human T1R3 was analysed from cell lysates obtained from THP‐1 cell line or PBMCs, as described.⁸⁶ Proteins in the culture supernatant were concentrated by chloroform‐methanol precipitation, as described previously.⁹⁰ Primary antibodies used were goat anti‐mouse IL‐1β antibody (R&D Systems, AF‐401‐NA; 1:1000), mouse monoclonal anti‐NLRP3 antibody (Adipogen, AG‐20B‐0014; 1:1000), mouse monoclonal anti‐caspase‐1 antibody (AdipoGen, AG‐20B‐0042; 1:1000), rabbit anti‐Atg7 antibody (Cell Signaling, 2631; 1:1000), rabbit anti‐human T1R3 (abcam, ab229015; 1:1000), and mouse anti‐β‐Actin antibody (BD Biosciences, 612657; 1:5000).