Ag 20b 0042 c100

Cell lysates and culture supernatants were incubated with cell lysis buffer (20 mM Tris–HCl [pH 7.4], 200 mM NaCl, 1% Nonidet P-40) and were denatured in lithium dodecyl sulfate sample buffer (M00676; GenScript) and boiled at 95°C for 10 min. SDS-PAGE–separated proteins were transferred to PVDF membranes and immunoblotted with primary antibodies against caspase-1 (AG-20B-0042-C100; Adipogen), IL-1β (GTX74034; Genetex), GSDMD (ab219800; Abcam), and β-actin (SC-47778HRP; Santa Cruz Biotechnology). Peroxidase-AffiniPure Goat Anti-Mouse (115-035-146; Jackson Immunoresearch Laboratories) or Anti-Rabbit (111-035-144; Jackson Immunoresearch Laboratories) secondary antibodies were used to detect proteins by enhanced chemiluminescence (34580; Thermo Fisher Scientific).

Cells and culture supernatants were incubated in cell lysis buffer (20 mM Tris HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P-40) for 10 min on ice followed by denaturation by boiling in Laemmli buffer for 10 min. Protein samples were resolved by SDS-PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes by semi-dry blotting. The PVDF membrane blocking, antibody incubation, and washing steps were performed using PBS containing 0.05% Tween 20 (v/v) together with 3% (w/v) non-fat dry milk. The incubation of the membranes with primary antibody was performed overnight at 4°C and the primary antibodies used in this study included: caspase-1 (AG-20B-0042-C100, 1:1,000, Adipogen), IL-1β (GTX74034, 1:3,000, GeneTex) and γ-Tubulin (T6557-100UL, 1:1,000, Sigma Aldrich). HRP-conjugated secondary anti-mouse and anti-rabbit antibodies (111-035-144 and 115-035-146; 1:5,000, Jackson ImmunoResearch Laboratories), followed by ECL (Thermo Scientific) incubation were used for signal detection and visualization.

For signaling blots, the supernatant was removed, and cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors plus 4× Laemmli sample buffer. Caspase and GSDMD cleavage were measured from the combined cell lysate and supernatants. Proteins were separated via SDS-PAGE with 8 to 12% polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore), and blocked with 5% nonfat dry milk. Primary antibodies against caspase-1 (AG-20B-0042-C100, Adipogen), caspase-3/cleaved-caspase-3 (9662 and 9661, CST), caspase-7/cleaved-caspase-7 (9492 and 9491, CST), caspase-8/cleaved-caspase-8 (4927, CST and AG-20T-0138-C100, Adipogen), caspase-9 (9504, CST), GSDMD (ab209845, Abcam), p-RIPK3 (91702, CST), RIPK3 (2283, ProSci), p-MLKL/MLKL (37333 and 37705, CST), and β-actin (4970, CST) were incubated overnight at 4°C followed by appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) incubated for 1 h at room temperature (Jackson ImmunoResearch, West Grove, PA). Membranes were visualized using Luminata Forte Chemiluminescence substrate (WBLUF0500, Millipore) or SuperSignal West Femto substrate (34096, Thermo Fisher Scientific) on a Bio-Rad ChemiDoc.

Lysis buffer containing 10 mM Tris-buffer (pH 7.6), 1% Triton X-100, 1% phosphatase inhibitor cocktail, and 1 mM PMSF was used to lyse cells. Cell lysates were then boiled in SDS sample buffer and resolved on a 10% SDS-PAGE gel. Immunoblots were incubated overnight with primary antibodies against NLRP3 (AG-20B-0014-C100, Adipogen, CH), ASC (AG-25B-0006, Adipogen, CH), mouse caspase-1 (AG-20B-0042-C100, Adipogen, CH), human caspase-1 (AG-20B-0048-C100, Adipogen, CH), IkBa (4814, Cell Signaling, USA), phospho-IkBa (2859, Ser32) (Cell Signaling, USA), A20 (23456-1-AP, Proteintech, USA), IL-1β (12242, Cell Signaling, USA) human GSDMD (sc-81868, Santa Cruz Biotechnology, USA), mouse GSDMD (ab209845, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, USA). Immunoblots were examined using an ECL detection reagent (Millipore Corporation, Billerica, MA, USA).

Primary microglial cells were plated on PDL‐coated coverslips. Cells grown on coverslips were washed with PBS and fixed in 4% PFA for 20 min at RT. The fixed cells were then washed with PBS and permeabilized in PBS containing 0.1% Triton X‐100 and 10% normal horse serum for 1 h. Cells were incubated with primary antibodies overnight at 4°C. After washing with PBS, the cells were incubated with Alexa Fluor 488‐ or 594‐conjugated anti‐rabbit or anti‐mouse IgG secondary antibodies for 1 h. Cell nuclei were stained with DAPI. The coverslips were imaged on a confocal laser microscope (ZEISS, Jena, Germany) with a 40× and 63× water‐immersion objective lens. The images were processed using the ZEN software. The primary antibodies used were anti‐caspase‐1 mouse antibody (1:250, AG‐20B‐0042‐C100, Adipogen), anti‐ASC rabbit antibody (1:500, NBP1‐78977, Novus), and anti‐TOM20 rabbit antibody (1:100, sc‐11415, Santa Cruz). Mitochondrial length was measured by tracing the fluorescence signals of TOM20 using the ZEN software developed by Carl Zeiss Microscopy. The average value of mitochondrial length in each group was measured and analyzed. For each group, approximately 300 mitochondria from three independent experiments were measured.

GPS (purity > 97%, #20831-76-9) was purchased from Yuanye Bio-Technology (Shanghai, China). GPS was dissolved in PBS as a stock solution at 1 g/ml and stored at 4 °C before use. Lipopolysaccharide (LPS) (L2630) was purchased from Sigma–Aldrich (USA). Antibodies against NLRP3 (AG-20B-0014-C100) and Caspase-1 (AG-20B-0042-C100) were purchased from Adipogen Life Sciences (San Diego, USA). Antibodies against ASC (#67824) and β-actin (#3700) were obtained from Cell Signaling Technology (Cell Signaling, Danvers, Technology, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse (SA00001-1), and HRP-conjugated goat anti-rabbit (SA00001-2) antibodies were purchased from proteintech (Boston, MA, USA). The BCA Protein Assay Kit was obtained from Beyotime (Beyotime Institute of Biotechnology, Nanjing, China).