The treated cells were lysed in RIPA buffer containing 1 mM DTT, 11 μg/ml DNase I, and protease inhibitor cocktail (Roche) and incubated on ice for 30 min. Sixty micrograms of protein sample were separated by electrophoresis on a denatured 10% SDS-PAGE gel and blotted onto a PVDF membrane (Millipore). The primary antibodies (Anti-Caspase-1, 1:1,000, ab138483, Abcam; Anti-cleavage Caspase-1, 1:1,000, ab207802, Abcam; Anti-Caspase-11, 1:1,000, ab22684, Abcam Anti-cleavage Caspase-11, 1:1,000, ab180673, Abcam) were incubated overnight at 4°C. Rabbit monoclonal to GAPDH (EPR16891, Abcam) was used as a loading control. Afterward, the membranes were rinsed and subsequently incubated with appropriate secondary antibodies. An ECL kit (Beyotime Biotechnology, Shanghai, China) was used to detect the bands of Western blots.
Ab138483
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab138483 is a rabbit monoclonal antibody. This antibody is designed to detect a specific target protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab214185, by Abcam (8 mentions)
Ab263899, by Abcam (5 mentions)
Ab219800, by Abcam (5 mentions)
Ab180673, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab22684, by Abcam (3 mentions)
Ab175449, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Ab209845, by Abcam (3 mentions)
Ab9722, by Abcam (3 mentions)
Ab207802, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Ab215203, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab16048, by Abcam (2 mentions)
Ab32536, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab109234, by Abcam (2 mentions)
36 protocols using ab138483
1
Western Blot Analysis of Caspase Activation
2
Immunofluorescence Assay for Apoptosis and Proliferation
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Fixation of cells with 4% formaldehyde was performed, and then they were washed three times using iced PBS. Next, cell permeabilization was achieved by using PBS with 0.25% Triton X‐100 and another three washes with iced PBS followed. Primary anti‐caspase‐1 antibody (Abcam, ab138483, 1:200), anti‐LC3II antibody (Abcam, ab192890, 1:200), and anti‐Ki67 antibody (Abcam, ab15580, 1:200) were added to the cells, and they were incubated overnight at 4°C. Then, secondary antibody (LS‐C60498, 1/1000, LSBio) was supplemented to the samples, and a 2‐h incubation at room temperature was done. Finally, the samples were mounted and imaged by a confocal microscope.
3
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).
4
Protein Expression Analysis in Cell Lines
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HK-2 cells and animal tissues were lysed using RIPA lysis buffer (Thermo Fisher Scientific, MA, USA). Each protein sample was electrophoresed by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Sigma-Aldrich). Next, the membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h and incubated with the following primary antibodies: anti-IRF2 (ab124744, 1:1000, Abcam); anti-Bax (ab32503, 1:1000, Abcam); anti-Bcl-2 (ab196495, 1:1000, Abcam); anti-caspase-1 (ab62698, ab138483, 1:1000, Abcam); anti-caspase-4 (4450, 1:1000, Cell Signaling Technology, MA, USA), anti-caspase-11 (ab246496, 1:1000, Abcam); anti- apoptosis-associated speck-like protein (ASC; 13833, 67824, 1:1000, Cell Signaling Technology); anti-TNF-α (3707, 1:500, Cell Signaling Technology); anti-IL-1β (12242, 1:500, Cell Signaling Technology); anti-IL-18 (54943, 57058, 1:500, Cell Signaling Technology); anti-IL-6 (ab259341, 1:500, Abcam); anti-GSDMD (39754, 1:1000, Cell Signaling Technology); anti-NLRP3 (13158, 15101, 1:1000, Cell Signaling Technology); and GAPDH (ab8245, 1:3000, Abcam) overnight at 4 °C. Each membrane was incubated with corresponding secondary antibodies (7233, 7076, 1:5000, Cell Signaling Technology) for 1 h at room temperature. Protein expression was detected using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific).
5
Western Blot Analysis of Inflammasome Proteins
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Proteins were drawn from the tissues and cells and quantified by using a BCA protein kit (Thermo Scientific). Proteins (50 μg) were loaded on SDS-PAGE gels per lane and transferred to PVDF membranes after electrophoresis. Then blocking membranes with 5% BSA at 2 h room temperature and incubating at 4°C overnight with the primary antibodies: GAPDH (Abcam, ab8245, 1:5,000), GSDMD (Santa Cruz Biotechnology, sc-393581, 1:1,000), caspase-11 (Abcam, ab22684, 1:1,000), caspase-1 (Abcam, ab138483, 1:1,000), caspase1 (p10), and caspase-1 (p20) (AdipoGen, AG-20B-0044, AG-20B-0042, 1:1,000). Immunoreactivity was detected by incubating with secondary antibodies (Abcam ab205718, ab97023, 1: 20,000).
6
Visualizing Colon Immune Responses
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Immunofluorescence staining of mouse colon sections was performed as previously described (26 (link)). The sections were incubated with primary antibodies against VDR (ab109234, Abcam), NLRP6 (ab58705, Abcam), Caspase-1 (ab138483, Abcam) and ASC (ab175449, Abcam) at 4°C overnight, followed by an incubation with fluorescently labelled secondary antibodies. The sections were examined using a Leica confocal microscope (LEICA TCS SP5).
7
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.
8
Molecular Profiling of Aortic Proteins
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Total proteins of aortas were extracted by RIPA buffer (Thermo Fisher, USA) and then subjected to SDS PAGE. After transfer, the PVDF membrane was blocked with 5% blotting grade blocker (Bio-rad, Hercules, CA, USA). Then, primary antibodies including anti-NLRP3 (1:1000 dilution, ab270449, Abcam, China), anti-caspase-1 p20 (1:1000 dilution, ab138483, Abcam, China), anti-β-actin (1:5000 dilution, ab179467, Abcam, China), anti-ICAM-1 (1:1000 dilution, ab222736, Abcam, China), anti-VCAM-1 (1:2000 dilution, ab134047, Abcam, China) was added and incubated for overnight at 4°C. Next day, secondary antibodies were incubated for 1 h at room temperature. The ECL Substrate (Thermo Fisher, USA) was added to visualize the bands. The band intensity was quantitated and analyzed using ImageJ.
9
Molecular Pathways of GSDMD-Mediated Inflammation
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Dimethyl sulfoxide (DMSO) (D8371, Solaibao, Beijing, China); Cell Counting Kit-8 (CA1210, Solaibao); BCA Protein Concentration Determination Kit (P0012S, Beyotime, Shanghai, China); RIPA buffer (R0010, Solaibao); Hematoxylin and Eosin Staining Kit (C0105S, Beyotime); 4% paraformaldehyde (BL539A, Biosharp, Hefei, China); 10% neutral buffer formalin fixative (Thermo right, Changchun, China); TMB Chromogen Solution (P0211, Beyotime); DAPI (C0065, Solaibao); Anti-GSDMD antibodies (ab219800, Abcam, Cambridge, UK); Anti-NLRP3 antibodies (ab214185, Abcam); Anti-caspase-1 antibodies (ab138483, Abcam); β-actin polyclonal antibody (20,536-1-AP, Proteintech, Rosemont, IL, USA); Anti-caspase-1 antibody (AB1871, Sigma,-Aldrich, USA); NLRP3 recombinant rabbit monoclonal antibody (sc06-23, Invitrogen, Waltham, MA, USA); Cy3-labeled goat anti-rabbit IgG (H + L) (A0516, Beyotime); Goat-anti-rabbit IgG (H + L) (A0208, Beyotime); Biotin-labeled Goat Anti-Rabbit IgG(H + L) (A0277, Beyotime); Empagliflozin (HY-15409, MedChemExpress, Princeton, NJ, USA); ECL Kit (Millipore, Billerica, MA, USA). CO2 constant temperature incubator (Thermo Fisher, USA); fluorescence microscope (DM500, Leica, Germany); Electrophoresis instrument electrophoresis system (Bio-Rad, Hercules, CA, USA); ChemiDoc™MP system (Bio-Rad).
10
Investigating Inflammatory Pathways in RAW264.7 Cells
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Uric acid (batch number BCBH0278V) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, United States). Colchicine (batch number 20190901) was purchased from Xishuangbanna Pharmaceutical Co., Ltd. Rat IL-1β (batch number MM0047R1) and TNF-α (batch number MM0180R1) ELISA kits were purchased from Meimian Co., Ltd. (Yancheng, China). Mouse TNF-α (batch number CSB-E04741m) ELISA kit was purchased from Cusabio Co., Ltd. (Wuhan, China). Primary antibodies against TLR2 (ab213676), TLR4 (ab217274), MyD88 (ab2064), NF-κB (ab76302), NLRP3 (ab214185), caspase-1 (ab138483), and IL-1β (ab9772) were purchased from Abcam Inc. (Cambridge, MA, United States). RAW264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
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