The brain was sectioned serially in 300 μm increments from the bregma to lambda, both sides of the PVN tissues were isolated by the use of a punch-out technique with a cryostat [33 (link), 34 (link)], and the PVN tissue was stored at − 80 °C until use. Western blotting analysis was performed in the same manner as previously described [6 (link)]. The protein levels were determined from tissue homogenate obtained from the PVN for the following antibodies: NLRP3 (1:2000, Santa Cruz, CA, USA), ASC (1:500, Santa Cruz, CA, USA), pro-caspase-1 (1:2000, Abcam, MA, USA), IL-1β (1:500, Santa Cruz, CA, USA), CXCR3 (1:2000, Abcam, MA, USA), VCAM-1, ICAM-1 (1:2000, Abcam, MA, USA), and CCL2 (1:2000, Santa Cruz, CA, USA), Iba-1 (1:500, Santa Cruz, CA, USA). The β-actin antibody was used as an internal standard, and band densities were analyzed with NIH ImageJ software.
Pro caspase 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Pro-caspase-1 is a protein involved in the activation of the inflammatory caspase-1 enzyme. Caspase-1 plays a key role in the processing and maturation of the pro-inflammatory cytokines IL-1β and IL-18. Pro-caspase-1 is the inactive precursor form of caspase-1 that requires cleavage for activation.
Automatically generated - may contain errors
Lab products found in correlation
Nlrp3, by Abcam (8 mentions)
Il 1β, by Abcam (6 mentions)
Pro il 1β, by Abcam (5 mentions)
Cleaved caspase 1, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
β actin, by Abcam (4 mentions)
Caspase 1, by Abcam (3 mentions)
Il 18, by Abcam (3 mentions)
Il 1β, by Cell Signaling Technology (3 mentions)
Nlrp3, by Cell Signaling Technology (3 mentions)
Gapdh, by Abcam (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Cell Signaling Technology (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
100 μl protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti mouse pro cleaved il 1β, by Cell Signaling Technology (2 mentions)
Ag 20b 0042, by Adipogen (2 mentions)
Kim 1, by Abcam (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Nlrp1, by Abcam (2 mentions)
31 protocols using pro caspase 1
1
Quantifying Inflammatory Markers in PVN
2
Western Blot Analysis of Inflammasome Markers
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Purified DCs (1×106) were lysed with 100-μl protein extraction reagent (89900, Thermo Scientific) containing protease inhibitor (5872S, Cell Signaling). Equal amounts of protein (lysate) or supernatants were run on an SDS-PAGE and transferred onto nitrocellulose membranes following electro blotting. After blocking with 5% fat-free milk, the membranes were incubated at 4°C overnight with the following primary antibodies: anti mouse pro & cleaved IL-1β (Cell Signaling, 12507), pro-caspase-1 (Abcam, ab108362), Caspase-p20 & 10 (Adipogen-AG-20B-0042) as per manufacturer's instructions. The membranes were then washed was incubated with Horseradish peroxidase–conjugated secondary antibody (Cell Signaling). Proteins were visualized with SuperSignal West Pico chemiluminescent substrate (34078, Thermo Scientific).
3
Protein Expression Analysis in Tissues
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After extracting the total protein from tissues and cellular samples using RIPA buffer with protease inhibitors, BCA quantitative measurements of protein concentrations were conducted. The relative expressions of the target proteins were normalized to β-actin. A total of 20 μg protein for each sample was separated by SDS-PAGE and transferred onto PVDF membrane (Cat No.: IEVH00005, Millipore, USA). After incubating with the milk in TBS for blocking, the membrane would be incubated the corresponding primary antibody, including anti-p16INK4a (Cat No.: ab108349, Abcam, UK), p21Waf/Cip1 (Cat No.: ab109520, Abcam, UK), SIRT1 (Cat No.: 2310, Cell Signaling, USA), NLRP3 (Cat No.: ab263899, Abcam, UK), apoptosis-associated speck-like protein (ASC, Cat No.: 13833, Cell Signaling, USA), pro-caspase-1 (Cat No.: ab179515, Abcam, UK), caspase 1 (Cat No.: ab207802, Abcam, UK) and β-actin (Cat No.: sc-47724, Santa Cruz, USA), at 4°C overnight. On the second day, the membrane was washed three times with TBST and incubated with the secondary antibody (Cat No.: sc-2357 and sc-2005, Santa Cruz, USA) for 1 h. The density of the bands was quantified using Labworks image acquisition software (UVP, USA) and was quantized using ImageJ software (NIH, USA).
4
Western Blot Analysis of Inflammatory Markers
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Samples were homogenized in lysis buffer, and the supernatant was extracted for measurement of total protein with a Protein Assay Kit (BCA; Pierce, Santa Cruz, CA, USA). Equal amounts of total protein were separated in SDS-PAGE and transferred to PVDF membranes in Trisglycine methanol buffer. The bands were visualized using the enhanced chemiluminescence. GADPH was developed as a loading control to normalize the data. The antibodies against NLRP3, ASC, pro-caspase-1, IL-1β, Kim-1 and IL-18 were purchased from Abcam (Cambridge, MA, USA). Antibody against BCL6 was obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NGAL and Cystatin C were purchased from Bioworld Technology, Ltd (Nanjing, Jiangsu, China). Antibody against GAPDH was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
5
Mitochondrial Fractionation and Western Blot Analysis
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The lysates were prepared using IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 10% glycerol) plus phosphatase and protease inhibitors (Roche, Mannheim, Germany). For mitochondrial fractionation, mitochondria were isolated from cytosolic components of the cells using a mitochondria isolation kit (Thermo Fisher, Rockford, IL, USA) according to the manufacturer’s protocols. Protein concentrations were determined by BCA assay and then lysates were subjected to SDS-PAGE, transferred onto 0.45 μm PVDF membrane (GE healthcare, Buckinghamshire, UK). The membranes were incubated with the appropriate primary antibodies for overnight. The primary antibodies used are as following: Caspase-3 (Rb, 1:1000; Cell Signaling, #9662), Cleaved Caspase-3 (Rb, 1:1000; Cell Signaling, #9664), Cytochrome C (Rb, 1:1000; Cell Signaling, #11940), Gasdermin D (Rb, 1:1000; Cell Signaling, #93709), PARP (Rb, 1:1000; Cell Signaling, #9542), p-STAT3 (Thy705) (Rb, 1:1000; Cell Signaling, #9145), DFNA5/GSDME (Rb, 1:1000; Abcam, ab215191), Pro-Caspase-1 (Rb, 1:1000; Abcam, ab179515), β-actin (Ms, 1:5000; Sigma, A5441). Immunoblots were detected using Amersham Imager 600 system (GE healthcare, Little Chalfont, UK). β-actin was used as an internal standard. At least three independent experiments were performed.
6
Western Blot Analysis of Inflammasome Markers
7
Protein Extraction and Western Blot Analysis
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Serum, urine, and kidney tissues were kept at sacrificed for protein extraction, and RIPA buffer was used for the sample suspension. The homogenate was centrifuged at 10,000 g for 30 min at 4 °C. 50 μg protein in mixture solution were separated with 10% SDS-PAGE and then transferred onto PVDF membranes of 0.45 μm (Millipore, Germany) incubated with 5% skimmed milk in phosphate-buffered saline at room temperature for 1 h (Li et al., 2019 (link)). After incubation with primary antibodies against and β-actin (Cell Signaling Technology, MA, United States) overnight at 4 °C, the membrane was incubated with secondary antibodies after washing with PBS and Tween 20. The target bands were detected with chemiluminescence. The antibodies against CCN1, NLRP3, ASC, pro-caspase-1, IL-1β, Kim-1, IL-18, NGAL, cystatin C, Bax, Bcl-2, and caspase-3 and caspase-9 were purchased from Abcam (Cambridge, MA, United States). All other chemicals were of analytical reagent grade.
8
Protein Extraction and Immunoblotting Protocol
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Total protein was extracted from cells or tissues using a whole protein extraction kit (AS1012, ASPEN). The primary antibodies (ASPEN) used were netrin-1 (1:1000), CD63 (1:2000), CD9 (1:2000), TSG101 (1:1000), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) (1:1000), apoptosis-associated speck-like protein (ASC) (1:1000), pro-caspase-1 (1:2000), cleaved caspase-1 (1:2000), pro-IL-1β (1:2000), IL-1β (1:2000) (Abcam), Unc5b (1:2000), PI3K (1:2000), p-PI3K (1:2000), Akt (1:2000), p-Akt (1:2000), mTOR (1:2000), p-mTOR (1:2000) and GAPDH (1:2000) (26,616, Thermo). A gel image processing system (gel Pro analyser software) was used to analyse the grey value of the target strip.
9
Investigating Inflammatory Signaling Pathways
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Chemical reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified. RPMI1640, fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Trypsin/EDTA was purchased from GE Healthcare Life Sciences (Logan, UT, USA). E-Cadherin rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AMPK-α rabbit polyclonal antibody, rabbit monoclonal antibodies specific to p-AMPK-α (Thr172), JAK2, p-JAK2 (Tyr1008), and occludin, and mouse monoclonal antibodies detecting STAT3, p-STAT3 (Tyr705), cleaved caspase 1, and IL-1β were purchased from Cell Signaling Technology Inc. (Boston, MA, USA). NOX2 and ICAM-1 rabbit monoclonal antibodies, and TGF-β, TNF-α, NOX1, p-p47phox, pro-caspase-1, and claudin 2 rabbit polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit polyclonal antibodies specific for IL-10 or IL-6 were from Abbiotec (San Diego, CA, USA), and NLRP3 rabbit polyclonal antibody was purchased from Novus Biologicals (Centennial, CO, USA). Tofacitinib citrate salt was obtained from L.C Laboratories (Woburn, MA, USA). VAS2870 was purchased from Sigma-Aldrich (St. Louis, MO, USA). BJ-3105 was synthesized by Byeong-Seon Jeong as reported [47 ].
10
Western Blot Analysis of Inflammatory Pathways
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The brain tissue and PBMCs were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche, Germany). Polyvinylidene fluoride membranes (Millipore, USA) were probed with primary antibodies against CD36 (1:4000; Abcam, catalog number: ab133625), NLRP3 (1:1000; Cell Signaling Technology, catalog number: 15101), ASC (1:1000; Cell Signaling Technology, catalog number: 67824), IL-1β (1:1000; Cell Signaling Technology, catalog number: 31202), Cleaved-IL-1β (1:1000; Cell Signaling Technology, catalog number: 83186), Caspase-1 (1:1000; Abcam, catalog number: ab207802), pro-Caspase-1 (1:1000; Abcam, catalog number: ab179515), NF-кB (1:1000; Cell Signaling Technology, catalog number: 8242), phospho-NF-кB (1:1000; Cell Signaling Technology, catalog number: 3033), CREB (1:1000; Cell Signaling Technology, catalog number: 9197), TrkB (1:1000; Cell Signaling Technology, catalog number: 4603), BDNF (1:1000; Cell Signaling Technology, catalog number: 47808) andβ-Actin (1:10,000; Abcam, catalog number: ab234437), overnight at 4 °C, and then incubated with secondary antibodies for 2 h. Signals were visualized with an ECL kit (Millipore, USA).
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