The total protein of GAS was extracted and quantified using a BCA protein quantification kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were loaded into 15 wells (20 μg total protein per well) of 4–12% Bis-Tris gradient gels (NP0336BOX, Invitrogen, Waltham, MA, USA). After separation by electrophoresis, proteins were transferred to nitro-cellulose Regular Stack (IB23001, Invitrogen). Membranes were blocked with Odyssey blocking buffer (LI-COR), and then incubated with anti-NLRP3 (CST, 15101S), anti-ASC (Novus Biologicals, NBP1-78977, Centennial, CO, USA), anti-Caspase-1 (CST, 2225S), anti-GSDMDC1 (Santa Cruz, sc-393656, Santa Cruz, CA, USA), anti-MuRF1 (Santa Cruz, sc-398608) and anti-Atrogin1 (Abcam, ab168372) antibodies overnight at 4 °C. Then, after being washed four times (8 min each) by Tris-buffered saline (TBS) containing Tween-20 (TBST), the membranes were incubated with goat anti-rabbit or anti-mouse IgG secondary antibodies (LI-COR, 926-68071/926-32210, Lincoln, NE, USA) at 25 °C for 1 h. Next, the membranes were washed with TBST four times (8 min each) and then TBS twice (5 min each). The protein bands were detected by a near-infrared spectroscopy detection system (Odyssey CLX, LI-COR). All bands were analyzed semi-quantitatively using Image Studio Ver 5.2 software [58 (link)].
Anti nlrp3
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
Anti-NLRP3 is a research-use-only antibody that specifically binds to the NLRP3 protein. NLRP3 is a key component of the inflammasome complex, which plays a crucial role in the innate immune response. This antibody can be used to detect and study the expression and localization of NLRP3 in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 1, by Santa Cruz Biotechnology (5 mentions)
Anti asc, by Santa Cruz Biotechnology (5 mentions)
Anti il 1β, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Anti myd88, by Santa Cruz Biotechnology (2 mentions)
Anti tlr4, by Santa Cruz Biotechnology (2 mentions)
Anti nf κb, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti caspase 1, by Novus Biologicals (2 mentions)
Anti nf κb, by Cell Signaling Technology (2 mentions)
Anti il 1β, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein quantification kit, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Np0336box, by Thermo Fisher Scientific (1 mentions)
Anti gsdmdc1, by Santa Cruz Biotechnology (1 mentions)
Anti murf1, by Santa Cruz Biotechnology (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Anti asc, by Enzo Life Sciences (1 mentions)
22 protocols using anti nlrp3
1
Quantitative Western Blot Analysis of NLRP3 Inflammasome
2
Inflammasome Colocalization in Mouse Kidney
3
Immunoblotting Analysis of Inflammation Markers
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Proteins (50 μg) from all samples were processed as previously described [28 (link)]. Membranes were incubated for 12 h at 4 °C with primary antibodies to anti-TLR4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MyD88 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NFκB (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NLRP3 (3 µg/mL; Novus, Dallas, TX, USA), anti-Caspase-1(1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-IL-1β (1 µg/mL; Thermo Fisher, Waltham, MA, USA) and β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Samples were maintained at room temperature for 30 min with peroxidase-conjugated secondary antibody diluted 1:1000 in 1 × Tris Buffered Saline (TBS), 5% milk and 0.05% Tween-20 (Signa-Aldrich, Milan, Italy) [29 (link)]. To visualize protein bands, the Electrochemiluminescence (ECL) method was used, and the protein levels were measured by means of the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA).
4
Western Blot for Protein Expression
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Western blot was performed as previously described.23 Proteins were loaded into SDS‐PAGE gel, and electrophoresis was performed. Then, the proteins were transferred to a nitrocellulose membrane, which was blocked with 5% non‐fat blocking grade milk (Bio‐Rad, Hercules) for 1 hour at room temperature. The membranes were incubated with the primary antibodies overnight at 4°C. The following primary antibodies were used: anti‐TXNIP (1:500; Santa Cruz Biotechnology), anti‐NLRP3 (1:1000; Novus Biologicals, Centennial), anti‐cleaved caspase‐1 (1:1000, Novus Biologicals), anti‐interleukin‐1β (1:1000; Santa Cruz Biotechnology), anti‐TNF‐α (1:500; Santa Cruz Biotechnology) and anti‐interleukin‐6 (1:1000; Santa Cruz Biotechnology). Anti‐β‐actin (1:3000; Santa Cruz Biotechnology) was used as loading control. The membranes were then incubated with appropriate secondary antibodies for 2 hours at room temperature. Immunoreactive bands were visualized with an ECL Plus kit (Amersham Biosciences) followed by exposure to X‐ray films and analysed using ImageJ software (NIH, Bethesda). For PC12 cells, expression of TXNIP and secreted pro‐inflammatory cytokines were measured from 4 separate experiments and normalized to expression of β‐actin in the cell lysate.24
5
Western Blot Analysis of Inflammatory Proteins
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Total proteins from tissues were extracted in RIPA lysis buffer (Solarbio, China), and a bicinchoninic acid (BCA) protein assay kit (Solarbio, China) was used for evaluating the protein concentration. The sample proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in TBST followed by incubating with primary antibody overnight. The primary antibodies were as follows: anti-RIPK3 (1:1,200; Novus Biologicals), anti-NLRP3 (1:400; Novus Biologicals), anti-caspase-1 (1:800; Novus Biologicals), anti- NF-κB/p65 (1:1,000; Cell Signaling Technology, CST), anti-β-actin (1:5,000; Proteintech Group, PTG), and anti-glyceraldehyde 3-phosphate dihydrogen (GAPDH) (1:5,000; Proteintech Group, PTG). After being washed with Tris Buffered Saline with Tween (TBST), the membranes were incubated with goat anti-rabbit second antibody for 1.5 h. Finally, the enhanced chemiluminescence (ECL; Thermo Scientific) was used to visualize immunoblots, and the densities of the relative target proteins were measured using ImageJ. The GAPDH or β-actin was chosen as the internal reference control.
6
Hippocampal Protein Extraction and Western Blotting
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The rat hippocampi were homogenized and placed in a radioimmunoprecipitation assay lysis buffer containing protease and phosphatase inhibitors and phenylmethylsulfonyl fluoride. The samples were lysed on ice for 30 minutes and centrifuged at 13,000 r/min for 10 minutes at 4°C, and the supernatant was aspirated. The bicinchoninic acid assay was performed to determine the protein concentration. The samples were then stored at −80°C until further use. Western blotting was performed using standard protocols [33 (link)]. The membranes were incubated overnight with primary antibodies, including anti-NLRP3 (1:500, Novus, USA), anti-GSDMD (1:1000, Abcam, UK), anti-caspase-1 (1:1000, Novus, USA), and anti-caspase-11 (1:200, Novus, USA), and then with the appropriate secondary antibodies. ImageJ software was used to measure the gray values of the target bands.
7
Antibodies for Glial Biomarkers Detection
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Antibodies for glial fibrillary acidic protein (GFAP), Nestin, and Caspase-1 were from Proteintech (Chicago, Illinois, USA). Antibodies against interleukin-1β (IL-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase conjugated antirabbit and mouse IgG were acquired from Cell Signaling Technology (Boston, Massachusetts, USA). Anti-Claudin-1 and anti-Synapsin-1 were purchased from Bioworld (Dublin, Ohio, USA), whereas anti-Occludin was from Thermofisher (USA). Anti-NLRP3 (nucleotide-binding domain-like receptor protein3) and anti-P2Y12 receptors were purchased from NOVUS (Cambridge, UK) and Abcam (Cambridge, UK), respectively.
8
Inflammatory Mechanisms in Hyperuricemia
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Allopurinol and carboxymethyl cellulose (CMC-Na) were purchased from Sigma-Aldrich (St. Louis, MO, USA). High-fructose corn syrup (90% fructose and 5% glucose) was purchased from Fonen and Fonher Enterprise Co. Ltd. (Tainan, Taiwan). Chrysin, a yellow powder and soluble in 0.5% CMC-Na solution, was purchased from Sigma-Aldrich (St. Louis, MO, USA), with a purity of ≥ 98%. The ELISA kit of xanthine oxidase (XO) was purchased from Cayman Chemical (Ann Arbor, MI, USA). The ELISA kit of IL-1β was purchased from BioLegend (San Diego, CA, USA). The rabbit polyclonal anti-ABCG2 and anti-OAT1 were obtained from Abcam (Cambridge, MA, USA). The rabbit polyclonal anti-GLUT9 was obtained from Millipore (Billerica, MA, USA), and anti-URAT1 was obtained from Proteintech (Rosemont, IL, USA). The mouse polyclonal anti-ASC and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit polyclonal anti-caspase-1 was obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-NLRP3 was obtained from NOVUS Biologicals (Littleton, CO, USA).
9
Immunofluorescent Analysis of Cardiac Tissue
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Hearts were resected and fixed in 4% paraformaldehyde for at least 1 day to prepare for immunofluorescence analysis. Thereafter, the specimens were soaked in gradient sucrose solutions in PBS to dehydrate and were processed into frozen sections. Representative sections from the ischemic region were permeabilized in 0.5% Triton X-100 for 15 min and sealed with 5% BSA for 1 h. Afterwards, the sections were incubated with the following primary antibodies at 4°C for 12 h: anti-COX2 (1:100, Cat no. ab15191; Abcam), anti-NLRP3 (1:100, Cat no. NBP2-12446; Novus, Littleton, CO, United States), anti-Vimentin (1:100, Cat no. 5741; CST) and anti-Smooth Muscle Actin (1:100, Cat no. sc-53142; Santa Cruz Biotechnology, Santa Cruz, CA, United States). Primary antibodies were detected by Alexa Fluor 488-conjugated goat anti-rabbit (1:500, Cat no. ab150077; Abcam), Alexa Fluor 568-conjugated goat anti-rabbit (1:500, Cat no. ab175471; Abcam), or Alexa Fluor 488-conjugated goat anti-mouse (1:500, Cat no. ab150113; Abcam) secondary antibodies, respectively, for 2 h at room temperature. Nuclei were identified with its marker of 4′,6-diamidino-2-phenylindole (DAPI) (Cat no. 62248; Thermo Fisher Scientific). Finally, the sections were observed with a fluorescence microscope.
10
Immunohistochemical Analysis of Pyroptosis
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To measure the impact of pyroptosis on POP, the uterine sacral ligament and vaginal wall were fixed in paraformaldehyde at 4 °C and incubated with the following primary antibodies overnight at 4 °C: anti-NLRP3 (1:400, NOVUS). After washing thrice with PBS, slides were incubated with secondary antibodies conjugated to Alexa fluor 488 (1:800, Invitrogen) at 37 °C for 1 h. The nuclei were labeled with DAPI, and fluorescent images were observed through a microscope.
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