Ab155970

The total protein from AC16 cells was extracted using a RIPA buffer (Solarbio, R0010, Beijing, China), and the protein level was determined by BCA protein assay kit (Thermo Fisher Scientific, PICPI23223). The sample was boiled at 95 °C for 10 min and separated through the gels with 10% concentration of SDS-PAGE gel (JRDUN Biotechnology Co., Ltd, Shanghai, China). Then, the separated proteins were transferred to polyvinylidene fluoride membrane and blocked with 5% nonfat milk. After blocking, the membrane was further incubated with anti-DUOX1, anti-active caspase-1, anti-GAPDH (67226-1-lg, 22915-1-AP, 60004-1-1G, Proteintech), anti-apoptosis-associated speck-like protein containing a CARD (ASC), anti-NLRP3, anti-pro-caspase-1, and anti-Gasdermin D-N domain (GSDMD-N) (Ab155970, Ab263899, Ab179515, Ab215203, Abcam) overnight at 4 °C with gentle shaking. After washing thrice by TBST(TBS + Tween20), the polyvinylidene fluoride membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime, Shanghai, China) for 1 h at 25 °C. Finally, the expression levels of proteins were measured using a chemiluminescent imaging system (Tanon 5200, Shanghai, China).

Ipsilateral basal cortical samples facing the blood clots were extracted. Western blot was performed as described previously^{37 (link)}. Briefly, cortical samples were homogenized, and centrifuged (1000 × g, 10 min, 4 °C). The supernatant was further centrifuged, and then, the protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). An equal amount of protein (50 μg) was suspended in loading buffer, then denatured at 95 °C for 5 min, and loaded on an SDS-PAGE gel. After electrophoresis and transfer onto polyvinylidene fluoride membranes, the membranes were blocked with non-fat dry milk buffer for 2 h and then incubated overnight at 4 °C with primary antibodies for LC3 (2775, 1:1000, Cell Signaling), Atg5 (ab54033, 1:500, Abcam), Parkin (ab15954, 1:1000, Abcam), PINK1 (ab75487, 1:500, Abcam), NLRP3 (ab98151, 1:800, Abcam), ASC (ab155970, 1:1000, Abcam), caspase-1 (ab1872, 1:1000, Abcam), IL-1β (SC-23460, 1:500; Santa Cruz), IL-18 (ab71495, 1:1000; Abcam). The membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein band densities were detected by X-ray film and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

ASC antibody (1:200, Abcam, ab155970, UK) was used for immunofluorescence in P. gingivalis-treated BMDMs (MOI = 50), with or without pretreatment of OTTSP167 or Compound 50 for 2 h. Mouse acet-tubulin antibody (1:500, Proteintech, 66,200-1-Ig, China) and mouse anti-α-tubulin (1:500, Proteintech, 66,031-1-Ig, China) were used for staining the structure of microtubuli. The color reactions of images were captured on the confocal microscopy (Nikon A1, Japan).

Gingival tissues were immediately stored at −80°C after excision. The tissues were cut into pieces, lysed with the RIPA lysis buffer (Beyotime, China), and homogenized with a homogenizer. BMDMs were lysed with the RIPA. Proteins were separated by SDS-PAGE (Smart-Lifesciences, China), transferred to a PVDF membrane (Millipore, USA), and blocked with QuickBlock™ blocking buffer (Beyotime, China). Rabbit anti-β-actin (1:1,000, CST, 4970 T, China), rabbit anti-NLRP3 (1:1,000, Affinity, DF7438, China), rabbit anti-caspase-1 (1:1,000, CST, 2225S, China), rabbit anti-MARK4 (1:800, Affinity, AF0693, China), rabbit anti-pro-IL-1β (1:1,000, Affinity, DF6251, China), rabbit anti-GSDMD (1:1,000, Proteintech, 20,770-1-AP, China), rabbit anti-cleaved-GSDMD (1:1,000, Abcam, ab255603, UK), mouse anti-α-tubulin (1:50,000, Proteintech, 66,031-1-Ig, China), rabbit anti-ASC (1:1,000, Abcam, ab155970, UK), mouse anti-acet-tubulin (1:50,000, Proteintech, 66,200-1-Ig, China), mouse anti-poly-tubulin (1:50,000, Proteintech, 66,375-1-Ig, China) and secondary antibodies (1:1,000, Thermo Fisher Scientific, USA) were used. The result of the color reactions was detected by ImageQuant LAS 4000.

The total protein was extracted from mouse kidney tissues or cells using radioimmunoprecipitation assay lysate (W063-1-1, Jiancheng) [34 (link)]. After the protein concentration was measured using the bicinchoninic acid kits (P0012S, Beyotime), the proteins (40 μg) were isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. Next, the membranes were blocked with 5% skim milk for 1 h and incubated with rabbit primary antibodies anti-Nephrin (1:1000, ab216341, Abcam), anti-Podocin (1:10000, ab181143), anti-Nrf2 (1:1000, ab92946), anti-heme oxygenase-1 (HO-1) (1:1000, ab68477), anti-NLRP3 (1:1000, ab270449), anti-ASC (1:5000, ab155970), anti-Pro Caspase-1 (1:200, ab238972), anti-Gasdermin D N-terminal domain (GSDMD-N) (1:1000, ab215203), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, ab9485) overnight at 4 °C. After washing with PBS, the membranes were incubated with HRP-labeled goat anti-rabbit secondary antibody IgG H&L (1:20000, ab97051) at room temperature for 30 min. After that, the membranes were developed by enhanced chemiluminescence and then observed and photographed. Image-Pro Plus 6.0 (Media Cybernetics, Inc., MD, USA) was adopted to analyze the relative expression of different proteins, with GAPDH as an internal reference.