Anti pro caspase 1

The protein extracts from the cortex and cultured astrocytes were homogenized in 250 mg tissue/0.5 ml cold lysis buffer (see above). Then they were kept on ice for 30 min, centrifuged at maximum speed for 15 min, and the supernatant was collected to determine the proteins levels using the Bradford Assay (Bio-Rad, Hercules, CA, USA). Lysates were separated by SDS-PAGE gels and were transferred to PVDF membranes following standard techniques. Membranes were blocked with 5% non-fat dried milk in TBS containing 0.1% Tween-20 (TBS-T). Next they were then incubated overnight with the following primary antibodies: anti-NLRP3 (1 μg/ml, Abcam), anti-caspase-1 (1/100, Santa Cruz Biotechnology); anti-pro-caspase-1 (1/200, Abcam); anti-Apaf-1 (1 μg/ml, Millipore Bioscience Research Reagents); anti-caspase-3 (1:500) and anti-caspase-9 (1:1000, Cell Signaling).
Some membranes were stripped for 1 h at 60°C in an SDS solution (2% SDS, 0.85% 2-ME, and 65 mM Tris-HCl, pH 6.8, and were washed and incubated with anti-GAPDH (1/3000, Chemicon) for 2 h as a loading control. The intensity of the bands was quantified with the image analysis program, α-Ease FC, version α Imager 2200 (Alpha Innotech Corporation).

Cells were prepared in 12-well plates and treated as described above. After incubation as indicated, supernatants were collected and concentrated using 20% (w/v) trichloroacetate, and the cells were lysed with 1 × SDS loading buffer (Beyotime, China) and then cell lysates were collected. Subsequently, concentrated supernatants and cell lysates were subjected to 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane by electroblotting. Next, the membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-IL-1β (R&D, USA), anti-caspase-1 p20 (AdipoGen, USA), anti-pro-IL-1β, anti-pro-caspase-1, anti-NEK7 (Abcam, Cambridge, UK) and anti-β-actin (Beyotime, Beijing, China). Finally, the distinct protein bands were detected by ECL detection reagent (Biosharp, China).

Proteins were extracted using Lysis Buffer (Tris-HCl 50 mM, NaCl 150 mM, EDTA 5 mM and Triton-X100 1%) and Cocktail Protease Inhibidor (04693159001, Roche). 14% polyacrilamide gelswere transferred to PVDF membrane using a semi-dry system. The membrane was blocked for 1 hour and incubated overnight at 4 °C with the primary antibody (Anti-pro-caspase-1, Gasdermin D, pro-IL-1β and ASC; Abcam). The membrane was then incubated for 1 hour with secondary antibody (Jacksons Immuno Research). The anti-β-actin antibody (Aldrich) was used as loading control. The bands were revealed using Chemiluminescent Substrate (Westar Supernova XLS3L and XLS3P) by Image Quant LAS 4000 (GE Healthcare Life Sciences). The bands were analyzed with the ImageJ software (Version 1.8) for densitometry analysis.

Western blot was described as described previously [23 (link)]. After rinsing the cells treated under indicated conditions with pre-chilled PBS solution, the cell pellets were resuspended in RIPA lysis buffer for 30 min for the extraction of total protein. Protein concentration was measured using a BCA protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Next, electrophoresis was performed to resolve the proteins with 10% SDS-PAGE at 120 V until the bromophenol blue dye front reached the separation adhesive base. The proteins were then transferred on to PVDF membranes (Millipore Sigma., MA, USA) in an icebox at 100 V for 1.5 h. The membranes were then blocked in blocking buffer for 1 h at 4°C. After washing with TBST solution, the membranes were incubated with primary antibodies including anti-pro-Caspase 1, anti-cleaved (CL)- Caspase 1, anti-GSDMD (1:1000; Abcam., MA, USA), and anti-GAPDH (1:3000, Leading Biology Inc., CA, USA) on a rocker at 4°C overnight followed by incubation with appropriate secondary antibodies (1:2000, MultiSciences, Shanghai, China) at room temperature for 2 h. Finally, the protein bands were visualized using an ECL detection system (Thermo Fisher Scientific, Inc., MA, USA).

Expression levels of pro‐caspase‐1, cleaved caspase‐1, YAF2, NLRP1 and adaptor apoptosis‐associated speck‐like (ASC) were determined using Western blot. Collected SH‐SY5Y cells were washed with 0.01 M PBS solution for twice. Then, the cells were broken using RIPA lysis solution (Solarbio) for protein extraction. Next, 20 μg of proteins for each group were separated on 12% SDS‐PAGE gels for 2.5 h, and were transferred into polyvinylidene fluoride membranes. After that, membranes were blocked with 5% fresh nonfat milk for 1 h followed by incubation with primary antibodies overnight at 4°C. The anti‐pro‐caspase‐1, anti‐ASC, anti‐NLRP1, anti‐ anti‐cleaved caspase‐1, and anti‐β‐actin (internal reference) antibodies were purchased from Abcam. All primary antibodies were diluted at a ratio of 1:2000 when used. Next day, membranes were maintained with secondary antibodies for 1 h at room temperature. Finally, an ECL kit was utilized to analyze protein bands.

The productions of interleukin 1 (IL-1β) and interleukin 6 (IL-6) were determined by the enzyme-linked immunosorbent serologic assay (ELISA) using commercially available kits (GBD) according to the manufacturer's instructions, after indicated treatments.
Western blotting was used to detect caspase-1, NLRP3, and NF-κB expressions. Briefly, the total cellular protein was extracted and separated by SDS-PAGE. SDS-PAGE was blotted to the PVDF membrane and incubated with anti-caspase-1 P20 (1 : 700; Abcam), anti-NF-κB (1 : 400; Abcam), anti-NLRP3 (1 : 600; Santa Cruz), anti-P-IκB-α (1 : 1000, Cell Signaling Technology), anti-procaspase-1 (1 : 800; Abcam), anti-cleaved IL-1β (1 : 700, Santa Cruz), and anti-IL-1β (full length) (1 : 1000, Abcam). The membranes were then incubated with the horseradish peroxidase-labeled secondary antibody, which was exposed to ECL color development reagents. The membranes were developed using the ChemiDoc-It™ TS2 Imaging System (Bio-Rad), and the relative optical density was analyzed using the ImageJ2x software (National Institute of Health, Bethesda, MD, USA).