Total protein was extracted from the cells by radioimmunoprecipitation assay (RIPA) lysate (AWB0136, Abiowell, China). Then, the protein was transferred to the polyvinylidene fluoride membrane after 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) treatment. The membrane was sealed with 5% skim milk (AWB0004, Abiowell) at room temperature for 2 h. AIM2 (1:1,500, 20590-1-AP, proteintech, Chicago, IL, USA), NLRC4 (1:1,000, ab201792, abcam, Cambridge, UK), NLRP3 (1:1,000, 19771-1-AP, proteintech), GSDMD-N (1:1,000, ab215203, abcam), ASC (1:2,000, 10500-1-AP, proteintech), caspase-1 (1:1,000, ab179515, abcam), IL-18 (1:8,000, 10663-1-AP, proteintech), IL-1β (1:1,000, 16806-1-AP, proteintech), and β-actin (1:5,000, 66009-1-Ig, proteintech) were incubated with the membrane at 4°C overnight. Then, the corresponding secondary antibodies were incubated with the membrane at room temperature for 2 h. The membrane was incubated with SuperECL Plus (AWB0005, abiowell), and then the protein bands were visualized by a chemiluminescence imaging system (ChemiScope 6100, Clinx, Shanghai, China).
Ab215203
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab215203 is a laboratory equipment product. It is designed for general use in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab263899, by Abcam (19 mentions)
Ab179515, by Abcam (11 mentions)
Ab207802, by Abcam (10 mentions)
Ab214185, by Abcam (8 mentions)
Ab9485, by Abcam (7 mentions)
Ab219800, by Abcam (6 mentions)
Ab6721, by Abcam (5 mentions)
Ab8227, by Abcam (4 mentions)
Ab254360, by Abcam (4 mentions)
Ab210070, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab151700, by Abcam (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (3 mentions)
Ab155970, by Abcam (3 mentions)
Ab1872, by Abcam (3 mentions)
Ab200478, by Abcam (3 mentions)
Ab207324, by Abcam (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
56 protocols using ab215203
1
Western Blot Analysis of Inflammasome Proteins
2
Western Blot Analysis of Protein Markers
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Cells were washed with PBS and lysed in lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin). The concentration of the protein was quantified using the BCA protein assay (Thermo Fisher Scientific, USA). Western blotting was implemented in accordance with the standard methods [47 (link)]. The antibodies used were as follows: PRMT5 (P0493, Sigma), GAPDH (HRP-60004, Proteintech), CASP1 (3866, CST), cleaved-CASP1 (4199, CST), H4R3me2s (ab5823, Abcam), cleaved-CASP3 (9661, CST), N-GSDMD (ab215203, Abcam), IL-1b (66737-1-Ig, Proteintech), and IL-18 (10663-1-AP, Proteintech). Quantifications of all western blotting signals with 3 repeated experiments are shown in Supplemental Fig. 2A–D as histograms.
3
Western Blot Analysis of Inflammasome Proteins
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Cells were prepared at 4°C in RIPA buffer (P0013J, Beyotime, China) containing proteinase inhibitor cocktail (B14001, Bimake, Houston, TX, USA) and phosphatase inhibitor cocktail (B15001, Bimake). Proteins were separated on 4%–20% precast mini polyacrylamide gels (SurePAGE™, GenScript, Nanjing, China) and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with anti-NEK7 (ab95873, Abcam), anti-GSDMD (ab219800, Abcam), anti-cleaved N-terminal GSDMD ab215203, Abcam), anti-NLRP3 (ab263899, Abcam), anti-cleavage caspase-1 (4199S, Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (9102S, Cell Signaling Technology), anti-p-ERK1/2 (9101S, Cell Signaling Technology), anti-α-SMA (A17910, ABclonal, Woburn, MA, USA), and anti-β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA). Membranes were then probed with appropriate secondary antibodies (Cell Signaling Technology). Immunoblot signals were detected by a Millipore chemical developer (Millipore Sigma, Burlington, MA, USA).
4
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Sigma, Shanghai, China) was applied to extract total proteins. A BCA kit (Sigma) was employed to measure the concentration of protein in the supernatant. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis was used to separate proteins, followed by their electrical transfer to a polyvinylidene fluoride membrane. Post blocking with 5% BSA, membranes were kept overnight at 4 °C alongside diluted primary antibodies: anti-USP11 (1:5000, ab109232, Abcam), anti-NLRP3 (1:1000, ab263899, Abcam, Cambridge, UK), anti-GSDMD-N (1:1000, ab215203, Abcam), anti-caspase-1 (1:1000, ab207802, Abcam), anti-IL-1β (1:1000, ab300501, Abcam), anti-IL-18 (1:1000, ab207323, Abcam), anti-p-IKKβ (1:1000, ab194528, Abcam), anti-IKKβ (1:500, ab32135, Abcam), anti-p-NF-kB (1:1000, ab76302, Abcam), anti-NF-kB (1:1000, ab16502, Abcam), anti-TRAF3 (1:1000, ab155298, Abcam), and anti-β-actin (1:1000, ab8227, Abcam) and then incubation for 1 h by secondary antibody. An ECL chromogenic substrate was used to visualize the bands.
5
Western Blot Analysis of NLRP3 Inflammasome Pathway
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Primary antibodies used in this study were anti‐NLRP3 (ab263899, 1:800; Abcam), anti‐ASC (ab175449, 1:500; Abcam), anti‐caspase‐1 (ab138483, 1 µg/mL; Abcam), anti‐N‐GSDMD (ab215203, 1:800; Abcam), anti‐inhibitor a of NF‐κB (IκBα) (#4814S, 1:800; Cell Signaling Technology), anti‐p‐IκBα (#2859, 1:800; Cell Signaling Technology), anti‐p65 (ab32536, 1:800; Abcam), LaminB1 (ab16048, 0.1 µg/mL; Abcam), and anti‐GAPDH (ab8245, 1:1000; Abcam). The nuclear and cytoplasmic proteins from RAW264.7 cells were isolated with a nuclear extraction kit (P0027; Beyotime). The total protein concentration in each cell lysate was determined with a BCA Protein Assay Kit (Beyotime). Subsequently, the proteins were separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 60 min and incubated with primary antibodies at 4°C overnight. Next day, the membranes were incubated with secondary antibodies (ab6721, 1: 2000; Abcam) at room temperature for 1.5 h. Finally, the protein expression was determined by an ECL kit and Scion Image v. 4.0.2 software (Scion Corporation).
6
Quantification of Autophagy and Inflammasome Markers
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After transfection, cells were collected and lysed. Then, total protein was collected and concentrated using a BCA kit. Protein (30 µg) was isolated using 10% SDS-PAGE at 120v. The protein was transferred onto PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies such as antiLC3I/II (ab62721, 1:2000, Abcam, USA), antiATG8 (ab98830, 1:2000, Abcam, USA), antiNLRP3 (ab263899, 1:1000, Abcam, USA), antiASC (ab283684, 1:1000, Abcam, USA), anti-Caspase1 (ab179515, 1:1000, Abcam, USA), GSDMD (ab215203, 1:1000, Abcam, USA), antiβ-actin (ab8227, 1:5000, Abcam, USA) and goat-anti-rabbit antibody (ab6721, 1:5000, Abcam, USA). Finally, the bands were captured by ECL reagents and analyzed using ImageJ (V.2.3.0).
7
Inhibition of DPP8/9 and Inflammasome Activation
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The small molecule inhibitor of DPP8/9, Talabostat mesylate (Val-boroPro mesylate), was purchased from MedChem Express (HY-13233A). LPS (InvivoGen tlrl-3pelps) was used at 1,000 EU/ml made in EGMV2 media. Nigericin was purchased from MedChem Express (HY-100381).
For cell-culture experiments, Talabostat was prepared in DMSO solution containing 0.1% TFA to prevent cyclization. TFA was purchased from Sigma-Aldrich (T6508-5Ml). Triton-X 100 was purchased from Merck (T8787-250 ml). The inhibitors Bortezomib (HY-10227), Rupintrivir (HY-106161), SNAP (82250-25), and Z-VAD-FMK (HY-16658B) were purchased from Cayman Chemical. Specific inhibitor Disulfuram (#PHR1690; Sigma-Aldrich) was purchased from Sigma, and Belcanasan (HY-13205) was purchased from MedChem Express.
Antibodies used were anti-NLRP1 (Cat.no AG-25B-0005-C100; Adipogen), anti-C-terminal–CARD8 (ab24186; Abcam), anti-N-terminal–CARD8 (ab194585; Abcam), anti-ASC (AG-25B-0006-C100; Adipogen), anti-pro-caspase-1+ p10 +p12 (ab719515; Abcam), anti-N-terminal–Gasdermin-D (ab215203; Abcam), cleaved GSDMD-NT (Asp275; 36425; Cell Signaling Technology), GSDMDC1 (NBP2-33422; Novus Biologicals), anti-eIF4G(C45A4) (2469; Cell Signaling Technology), anti-PABP (10910-1-AP; Proteintech), anti–IL-18 (ab201324; Abcam), anti–IL-1β (AB201; R&D systems), and anti–β-actin (A2228-100 µl; Sigma-Aldrich).
For cell-culture experiments, Talabostat was prepared in DMSO solution containing 0.1% TFA to prevent cyclization. TFA was purchased from Sigma-Aldrich (T6508-5Ml). Triton-X 100 was purchased from Merck (T8787-250 ml). The inhibitors Bortezomib (HY-10227), Rupintrivir (HY-106161), SNAP (82250-25), and Z-VAD-FMK (HY-16658B) were purchased from Cayman Chemical. Specific inhibitor Disulfuram (#PHR1690; Sigma-Aldrich) was purchased from Sigma, and Belcanasan (HY-13205) was purchased from MedChem Express.
Antibodies used were anti-NLRP1 (Cat.no AG-25B-0005-C100; Adipogen), anti-C-terminal–CARD8 (ab24186; Abcam), anti-N-terminal–CARD8 (ab194585; Abcam), anti-ASC (AG-25B-0006-C100; Adipogen), anti-pro-caspase-1+ p10 +p12 (ab719515; Abcam), anti-N-terminal–Gasdermin-D (ab215203; Abcam), cleaved GSDMD-NT (Asp275; 36425; Cell Signaling Technology), GSDMDC1 (NBP2-33422; Novus Biologicals), anti-eIF4G(C45A4) (2469; Cell Signaling Technology), anti-PABP (10910-1-AP; Proteintech), anti–IL-18 (ab201324; Abcam), anti–IL-1β (AB201; R&D systems), and anti–β-actin (A2228-100 µl; Sigma-Aldrich).
8
Protein Expression Analysis in HK-2 Cells
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Total protein was extracted from HK-2 cells using RIPA lysis buffer containing protease inhibitor cocktail (Thermo Fisher Scientific, MA, USA). Cells suspended in RIPA buffer were lysed on ice for 10 min and the cell lysate was centrifuged at 12,000 × g for 10 min. Protein concentration was quantified by a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Fifteen micrograms of protein was used for SDS-PAGE electrophoresis, and separated proteins were transferred onto the PVDF membrane. After blocking with 5% skimmed milk for 1 h, the membrane was then incubated with primary antibodies (1:1000 dilution, all from Abcam), including NLRP3 (ab263899), Caspase-1 (ab207802), IL-1β (ab254360), GSDMD-N (ab215203), SGK1 (ab32374), and GAPDH (ab8245). After washing with TBS Tween 20, membranes were subject to further probing using HRP-linked secondary antibody (1:3000; Cell signaling technologies, MA, USA). An ECL chemiluminescence kit (Thermo Fisher Scientific, MA, USA) was employed to detect protein bands, and GAPDH was used for internal reference for protein quantification using ImageJ software (Bethesda, MD, USA).
9
Western Blot Analysis of NLRP3 Inflammasome
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VSMCs in the logarithmic phase were collected, and the total protein in the cells was extracted by RAPI lysate to detect the protein concentration. The protein was isolated by 10% SDS-PAGE and the target band was transferred to PVDF membrane by electroporation. After blocking with 5% skim milk powder at room temperature for 1 h, primary antibodies were added, including HMGB1(ab18256,1:5000, Abcam) and GSDMD (ab219800,1:1000, Abcam), N-GSDMD (ab215203,1:5000, Abcam), ASC(ab151700,1:1000, Abcam), NLRP3 (ab263899,1:1000, Abcam), Caspase-1(ab138483,1:5000, Abcam), Cleaved-Caspase-1(#404991:1000, SAB), IL-6 (21865-1-AP,1:1000, proteintech), TNF-α (17590-1-AP,1:1000, proteintech), IL-1β (16806-1-AP,1:5000, proteintech) and GAPDH (60004-1-IG,1:1000, proteintech) were incubated overnight at 4 °C. After that, the HRP-conjugated Affinipure Goat Anti-Mouse IgG (SA00001-1, 1:50000, proteintech) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (SA00001-2, 1:1000, proteintech) was added and incubated at room temperature for 2 h. Finally, ECL staining was performed and images were collected by a gel imager.
10
Protein Extraction and Western Blot Analysis
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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).
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