The target cell protein was obtained by RIPA method, and the protein concentration was determined by BCA kit (Solarbio, Beijing, China). Then, prepared protein was taken for gel electrophoresis separation, and the isolated protein was electrically transferred to PVDF membrane. After blocked with 5% skim milk, the PVDF membrane was subjected to primary antibodies: connective tissue growth factor (CTGF, #ab6992, 1:1000, Abcam, Cambridge, UK), collagen I (#ab270993, 1:1000, Abcam, Cambridge, UK), collagen III (#ab7778, 1:1000, Abcam, Cambridge, UK), Bcl-2 (#ab196495, 1:1000, Abcam, Cambridge, UK), Bax (#ab32503, 1:1000, Abcam, Cambridge, UK), NOD-like receptor protein 3 (NLRP3, #ab263899, 1:1000, Abcam, Cambridge, UK), ASC (rat: #ab180799, 1:1000, Abcam, Cambridge, UK; mouse: #67824S, Cell Signaling, Danvers, MA), caspase-1 (#83383, 1:1000, Cell Signaling, Danvers, MA), IL-β (#ab254360, 1:1000, Abcam, Cambridge, UK), IL-18 (#ab191860, 1:1000, Abcam, Cambridge, UK) and GAPDH (#ab9485, 1:1000, Abcam, Cambridge, UK). The next day, the PVDF membrane was incubated with secondary antibody (#ab288151, 1:5000, Abcam, Cambridge, UK) at room temperature. An enhanced chemiluminescence kit (ECL, #P0018S, Beyotime Biotechnology, Beijing, China) was added and exposed in the gel imaging system. The protein content was analysed using Quantity-One software (Bio-Rad, Hercules, CA).
Ab191860
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab191860 is a rabbit monoclonal antibody that recognizes the protein target. It is intended for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab263899, by Abcam (5 mentions)
Ab214185, by Abcam (5 mentions)
Ab219800, by Abcam (3 mentions)
Ab254360, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Ab283818, by Abcam (2 mentions)
Af4022, by Affinity Biosciences (2 mentions)
Ab8245, by Abcam (2 mentions)
Ly294002, by Merck Group (2 mentions)
Ab6671, by Abcam (2 mentions)
Ab234437, by Abcam (2 mentions)
Ab1872, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Df6304, by Affinity Biosciences (2 mentions)
Df7438, by Affinity Biosciences (2 mentions)
Ab32536, by Abcam (2 mentions)
Ab270993, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab180799, by Abcam (1 mentions)
16 protocols using ab191860
1
Western Blot Analysis of Protein Markers
2
Immunoblotting for Oxidative Stress Markers
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Immunoblotting was performed as described previously [4 (link),19 (link)]. The primary antibodies were used as follows: transforming growth factor-beta1 (TGF-β1, #ab179695, Abcam), connective-tissue growth factor (CTGF, #ab6992, Abcam), pro-interleukin-1beta (pro-IL-1β, #ab9722, Abcam), pro-IL-18 (#ab191860, Abcam), NOD-like receptor pyrin domain-containing protein 3 (NLRP3, #ab214185, Abcam), tumor necrosis factor-alpha (TNF-α, #ab205587), inducible nitric oxide synthase (iNOS, #ab178945), superoxide dismutase-2 (SOD2/MnSOD, #ab13534, Abcam), thioredoxin (TRX, #ab109385), thioredoxin-interacting protein (TXNIP, #ab188865), Parkin (#2132, Cell Signaling Technology), succinate dehydrogenase complex subunit A (SDHA, #ab66484, Abcam), C/EBP homologous protein (CHOP, L63F7, #2895, Cell Signaling), inositol-requiring protein-1α (IRE-1α, phospho S724, #ab37073, Abcam), B-cell lymphoma-2 (Bcl-2, #ab196495, Abcam), Bcl2-associated X (Bax, #ab32503, Abcam), p-JNK (#ab76572), p-MEK4 (#4514, Cell Signaling), p-ASK1 (#ab278547), p-P38 (#ab4822), p-MEK3 (#ab79586), beta actin (β-actin, #ab8226, Abcam). Images were analyzed with an image analyzer (Odyssey® CL Imaging System, LI-COR Biosciences, NE, USA). Optical densities were obtained using the sham group as 100% reference and normalized with β-actin.
3
Western Blot Analysis of Inflammasome Proteins
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After choosing RIPA buffer (Solarbio, R0010) to lyse cells, we then centrifuged the turbid liquid and collected the supernatant. Protein samples were quantified to 20 μg by a BCA protein assay kit (Solarbio, PC0020), then loaded in SDS-PAGE for further separation and later transferred onto PVDF membranes. The PVDF membranes were blocked with 5% fat-free milk at room temperature for 1 h. After being washed 3 times, the membranes were soaked in primary antibodies against GSDMD (Santa Cruz, sc-393581, 1 : 200), MyHC (Abcam, ab50967, 1 : 1000), NLRP3 (Abcam, ab263899, 1 : 1000), IL-1β (Abcam, ab283818, 1 : 1000), and IL-18 (Abcam, ab191860, 1 : 1000) at 4°C overnight. The membranes were subsequently reprobed with GAPDH (Cell Signaling, 2118S, 1 : 1000) for standardization. After being washed three times, they were incubated in anti-mouse IG-HRP (Beyotime, A0216) or anti-rabbit IG-HRP-(Beyotime, A0208) secondary antibodies for 1 h. Finally, visualizations of proteins by High-sig ECL Substrate (Bio-Rad, USA) were analyzed with ImageJ software.
4
Protein Expression in Ischemic Penumbra
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The rats were sacrificed using sodium pentobarbital after three days of treatment with EP, and then the ischemic penumbra was collected as described previously [17 (link)]. According to the manufacturer’s instructions, tissue lysate was measured using the BCA protein assay kit (PC0020, Solarbio). Thereafter, the lysates were normalized to equal amounts per group, and 10 μg protein from the tissue lysates was split in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and transferred to PVDF membrane with 1% blocking buffer (C1090, Beyotime) for 1 h at room temperature. Then, the membranes were incubated with primary antibodies, including NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab17544, Abcam), GSDMD (1:1000, ab219800, Abcam), Cleaved-Caspase 1 (1:1000, #AF4022, Affinity), IL-1β (1:1000, ab254360, Abcam), IL-18 (1:1000, ab191860, Abcam), β-actin (1:5000, ab8227, Abcam), and GAPDH (1:5000, ab8245, Abcam) overnight at 4 °C. Thereafter proteins were determined using the appropriate species-specific HRP-conjugated secondary antibodies for 1 h at room temperature. Representative bands were visualized by ECL WB reagents, and the relative band optical intensity was quantified using imagej.js. The intensity for each protein was normalized to that evaluated for β-actin or GAPDH and was expressed as the relative fold to the sham group.
5
Piperine Modulates PI3K/AKT and NLRP3 Pathways
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Piperine (PIP) specified to be more than 97% pure was purchased from Shanghai Winherb Medical Science Co. (Shanghai, China).13 Lactate dehydrogenase (LDH) and creatine kinase (CK) were detected by commercially available ELISA kits (Jiancheng Bioengineering Institute). The following primary antibodies were from Abcam: anti‐p‐PI3K (1:600 dilution, ab182651), anti‐t‐PI3K (1:400, ab191606), anti‐NLRP3 (1:1000, ab263899), anti‐IL‐18 (1:500, ab191860) and anti‐GAPDH (1:1000, ab37168). The following primary antibodies were obtained from Cell Signaling Technology: anti‐p‐AKT (1:800, #9145), anti‐t‐AKT (1:600, #9139), anti‐cleaved caspase‐1(1:600, #89332), anti‐pro‐caspase‐1 (1:1000, #24232), anti‐cleaved IL‐1β (1:500, #63124) and anti‐pro‐IL‐1β (1:500, #12703). Antibody against RP105 (1:600, PAB18126) was obtained from ABNOVA. The BCA protein assay kit was purchased from Pierce. Evans blue and TTC dying were purchased from Beyotime Institute of Biotechnology. LY294002 (a PI3K/AKT inhibitor) were from Sigma‐Aldrich. The miR‐383 mimic and its scrambled oligonucleotides (miR‐NC) were produced by GenePharma Co., Ltd.
6
Inflammatory Bowel Disease Pathways
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2,4,6-trinitrobenzenesulfonic acid (TNBS; P2297, Sigma), anti-IL-18 (ab191860, Abcam), anti-TNF-α (ab6671, Abcam), anti-NF-κB p65 (ab16502, Abcam), anti-microtubule-associated protein 1 Light chain 3 beta (LC3B; #3868, Cell Signaling Technology, CST), anti-sequestosome 1 (p62; #23214, CST), anti-Beclin-1 (#3738, CST), anti-phospho-mTOR (p-mTOR; #2971, CST), Cy3-AffiniPure goat anti-mouse IgG (H + L) (111-165-003, Jackson), HRP-labeled goat anti-rabbit IgG (H + L) (A0208, Beyotime), HRP-labeled goat anti-mouse IgG (H + L) (A0216, Beyotime), insulin (P3376, Beyotime), rapamycin (LC R-5000, LC Laboratories), anti-PI3K p85 (#4292, CST), anti-Akt (#9272, CST), anti-p-Akt (#9271, CST), anti-Vps34 (V9764, sigma), and Antifade mounting medium (Beyotime, P0126) were used in this study.
7
Western Blot Analysis of NLRP3 Inflammasome
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Lysates from tissues and cells were prepared using RIPA lysis buffer and were quantified using a BCA kit (cat. #BCA1; Sigma–Aldrich). Afterwards, lysates were resolved in SDS-PAGE to separate proteins and were then transferred to polyvinylidene fluoride membranes. The membranes were immersed in 5% skimmed milk to block for non-specific binding and labelled with anti-WHSC1 (1:2000; cat. #ab223694; Abcam), NLRP3 (1:500; cat. #ab214185; Abcam), ASC (1:2000; cat. #ab175449; Abcam), caspase-1 p20 (1:1000; cat. #PA5-99390; Thermo Fisher Scientific), IL-18 (1:1000; cat. #ab191860; Abcam), IL-1β (1:1000; cat. #ab234437; Abcam), GSDMD FL (full length GSDMD; 1:1000; cat. #ab219800; Abcam), GSDMD CL (GSDMD-N terminal segment; 1:1000; cat. #10137S; Cell Signaling Technology, Danvers, MA), NEK7 (1:3000; cat. #ab133514; Abcam) and β-actin (1:5000; cat. #ab8227; Abcam) Abs at 4°C for 12 h. Afterwards, the membranes were incubated with IgG H&L (HRP; 1:5000; cat. #ab6728; Abcam) for 2 h at room temperature. β-Actin was designated as the internal control protein. Densitometry of bands was measured utilising the ECL Western Blotting Substrate (cat. #32132X3; Thermo Fisher Scientific).
8
Macrophage Polarization and Inflammatory Signaling
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Synthetic human IMD1–53, rat IMD17–47 and AngII were from Phoenix Pharmaceuticals (Belmont, CA, USA). Alzet Mini-osmotic pumps (Alzet model 1004) were from DURECT Corp. (Cupertino, CA, USA). DAPT, LPS, IFN-γ, IL-4, PI3K/Akt inhibitor LY294002, cAMP/PKA inhibitor H89 and cGMP/PKG inhibitor Ly83583 were from Sigma (MO, USA). Primary antibodies for Notch1 (ab52627), hes1 (ab71559), NICD (ab8925), iNOS (ab3523), NLRP3 (ab214185), caspase-1 (ab1872), CD68 (ab955), CD16 (ab203883), Arg1 (ab91279), CD206 (ab64693, ADAM10 (ab1997), ADAM17 (ab2051), IL-1β (ab2105), IL-10 (ab9969) and IL-18 (ab191860) were from Abcam (Cambridge, UK). Primary antibodies for p-Akt (Ser473) (4060) and Akt (9272) were from Cell Signaling Technology(CST, USA). Primary antibodies for β-actin (sc-1616), GAPDH (sc-25778), ASC (sc-514414), and all secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and reagents were of analytical grade. Mouse macrophage line Raw264.7 were bought from American Type Culture Collection (ATCC, USA).
9
Western Blot Analysis of Inflammatory Markers
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Cells were lysed in RIPA Lysis Buffer (Beyotime, Haimen, China). Lysates were then separated on a 10% SDS polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% milk for 1 h and incubated overnight at 4°C with primary antibodies. Primary antibodies, including anti-IL12A antibody (1:1,000, ab133751), anti-IL18 antibody (1:2,000; ab191860), anti-NF-κB p50 (1:3,000, ab32360), and anti-GAPDH (1:500, ab22048), were purchased from Abcam (Cambridge, MA, USA). After rinsing, immunocomplexes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:2,000, Zhongshanjinqiao, Beijing, China) for 30 min at 37°C. Immunoreactive bands were visualized using an enhanced chemiluminescence system (ECL; GE Healthcare, Madrid, Spain).
10
Western Blot Analysis of Inflammasome Pathway
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RSC96 cells were seeded in 10-cm dish (2 × 105 cells) and treated with 25-mM HG, 1-μM loganin or both. Then, cells were lysed in M-PER (mammalian protein extraction reagent, 78501, Thermo Fisher Scientific, Waltham, MA, USA) containing EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitor (Roche Diagnostics, Mannheim, Germany). The lysates were separated by SDS-PAGE (7.5–12%) transferred to PVDF membrane in blocking buffer (TBST containing 3% bovine serum albumin) and incubated with primary antibodies at 4 °C overnight. Purified mouse anti-rat β-actin (A5441, Sigma-Aldrich, St. Louis, MO, USA) was used to normalize the signals generated from anti-phospho-NF-κB (#3033, Cell Signaling, Danvers, MA, USA), NF-κB (#6956, Cell Signaling), NLRP3 (ab214185, Abcam, Cambridge, UK,), ASC (SAB4501315, Sigma-Aldrich), caspase-1 (A0964, ABclonal, Woburn, MA, USA), IL-1β (ab9722, Abcam), IL-18 (ab191860, Abcam), GSDMD (#93709, Cell Signaling) and TXNIP (#14715, Cell Signaling). The membrane was washed with 1× TBS-T (TBS containing 0.1% Tween-20) and the bound antibodies were visualized by developing with ECL western blotting detection reagents (#WBKLS0500, Millipore, Temecula, CA, USA).
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