Aliquots of protein extracts (20 μg) derived from THP1 monocytes, their derived macrophages, HUVECs, and HASMCs were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then immunoblotted with specific antibodies raised against the following proteins: CD68, ACAT1, ICAM1, VCAM1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ABCA1, collagen 1 (Novus Biologicals, Littleton, CO, USA), collagen 3, fibronectin, arginase 1, phospho (Ser529)-NFκB, α-tubulin, MMP2 (GeneTex, Irvine, CA, USA), MMP9 (EnoGene, Atlanta, GA, USA), elastin, MARCO, selectin E (Bioss, Woburn, MA, USA), PPARγ (Signalway Antibody, College Park, MD, USA), phospho (Thr202/Tyr204)-ERK1/2, phospho (Ser/Thr)-Akt (Cell Signaling Technology, Tokyo, Japan), c-Src (Bioworld Technology, St. Louis Park, MN, USA), PI3K, Bcl2, Bax, and β-actin (Sigma). The band intensity of the immunoblot was quantified by densitometry [39 (link),40 (link),41 (link),44 (link),45 (link),46 (link),47 (link),48 (link),49 (link)].
Arginase 1
Manufactured by GeneTex
Sourced in United States
Arginase-1 is an enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. It plays a crucial role in the urea cycle, a metabolic pathway responsible for the conversion of toxic ammonia into less toxic urea. Arginase-1 is an important marker for the identification and study of liver cells and their function.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
Vcam 1, by Santa Cruz Biotechnology (2 mentions)
α tubulin, by GeneTex (2 mentions)
Icam 1, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Elastin, by Bioss Antibodies (1 mentions)
Acat1, by Santa Cruz Biotechnology (1 mentions)
Abca1, by Novus Biologicals (1 mentions)
Collagen 1, by Novus Biologicals (1 mentions)
Collagen 3, by GeneTex (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
C src, by Bioworld Technology (1 mentions)
Bcl 2, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Merck Group (1 mentions)
Pparγ, by Signalway Antibody (1 mentions)
Fibronectin, by GeneTex (1 mentions)
Phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
Cox 2, by Cayman Chemical (1 mentions)
Marco, by Bioss Antibodies (1 mentions)
Nf κb, by Aviva Systems Biology (1 mentions)
7 protocols using arginase 1
1
Protein Expression Profiling of Vascular Cells
2
Macrophage Isolation and Immunoblotting
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Peritoneal cells were immediately suspended in RPMI-1640 medium supplemented with 10% FBS, 0.05 mg/ml streptomycin, and 50 U/ml penicillin, and were seeded onto 6-cm dishes (4 × 106 cells per 2 ml/dish). After incubation for 1 h at 37°C in 5% CO2 to allow adhesion, the medium was discarded to remove nonadherent cells. Adherent macrophages were immediately harvested after washing the dishes with PBS twice for immunoblotting with antibodies to pentraxin-3, MARCO (Bioss), arginase-1, MCP-1, JNK (GeneTex), COX-2 (Cayman Chemical, Ann Arbor, Michigan), NF-κB (Aviva Systems Biology), phosphorylated ERK1/2 (Cell Signaling Technology), or β-actin (Sigma) 19 (link), 22 (link).
3
Immunohistochemical Analysis of Neuroinflammation
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Primary antibodies were used against Iba-1 (mouse monoclonal IgG, 1:500 (Millipore) or rabbit polyclonal IgG, 1:1000 (Wako)), inducible nitric oxide synthase (iNOS) (rabbit polyclonal IgG, 1:200 (Abcam)), CD68 (mouse monoclonal IgG, 1:500 (Bio-Rad)), Arginase-1 (rabbit polyclonal IgG, 1:2000 (Gene Tex)), CD206 (goat polyclonal IgG, 1:400 (R &D systems)), Ki-67 (rabbit polyclonal IgG, 1:2000 (Novocas)), and GFAP (mouse monoclonal IgG, 1:10000 (Millipore)). For detection of the primary antibodies, Alexa488 or Alexa594-conjugated secondary antibodies (anti-rabbit or mouse IgG, 1:2000 (Thermo Fisher)) were used. Vessel walls were visualized by Alexa594-conjugated phalloidin (1:500, (Thermo Fisher)).
4
Immunoprecipitation of PP2A and Arginase-1
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Immunoprecipitation experiments were performed using SureBeads™ Protein A/G Magnetic Beads (BioRad SureBeads™ Protein G #161-4023). Briefly, 100 μl SureBeads were magnetized and washed with a solution of PBS/Tween20 0.1% v/v., then incubated for 30 min with 2 μg anti- PP2A (Genetex # GTX106334, Irvine, CA, USA) and Arginase-1 (Genetex # GTX109242, Irvine, CA, USA) at room temperature. After incubation, beads were washed 3 times with PBS/Tween20 0.1% v/v and the beads/antibody complexes were recovered and incubated with 100 μg of protein extracts for 1 h at room temperature. Proteins were separated by SDS-PAGE followed by immunoblotting on a nitrocellulose membrane (Bio-Rad, Hercules CA, USA). Membrane were incubated with the antibodies anti-HNE (1:2000, Novus Biologicals, Abingdon, UK), and then detected by the peroxidase-conjugated secondary antibody (1:20.000, Bio-Rad, Hercules, CA, USA) with Clarity™ Western ECL Blotting Substrates (Bio-Rad, Hercules, CA, US). Membranes were then acquired with ChemiDoc MP image system (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab software (Bio-Rad, Hercules, CA, USA).
5
Protein Expression Analysis in Vascular Cells
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Aliquots of protein extracts derived from THP-1 monocytes, THP-1-derived macrophages, HASMCs, and HUVECs were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then immunoblotted with antibodies raised against the following proteins: CD36, CD68, ACAT-1, ICAM-1, VCAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ABCA1, phosphorylated nuclear factor-κB (p-NF-κB), phosphorylated c-jun N-terminal kinase (p-JNK), α-tubulin, arginase-1 (GeneTex, Irvine, CA, USA), E-selectin, MARCO (Bioss, Woburn, MA, USA), peroxisome proliferator-activated receptor-γ (PPAR-γ; Signalway Antibody, College Park, MD, USA), phosphorylated Akt, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated p38 (p-p38), Bax (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (Abcam, Cambridge, UK), cleaved caspase-3 (R&D Systems, Minneapolis, MN, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Acris-OriGene Technologies, Herford, Germany), and β-actin (Sigma, St. Louis, MO, USA) [2 (link), 29 (link)–40 (link)]. Proteins were visualized by enhanced chemiluminescence western blotting detection reagents (GE Healthcare, Amersham, UK).
6
Retinal Protein Expression Analysis
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Retinas were collected, snap-frozen, and stored at −80 oC until analysis. Frozen retinas or cells were homogenized in RIPA buffer (Thermofisher Scientific). Western blotting was conducted as previously described [2 (link)]. The primary antibodies used were IL-1β (R&D Systems, Cat. # AF-401-NA), Iba-1 (FUJIFILM Wako, Cat. # 019-19741), p-NF-κB (Cell Signaling, Cat. # 3033), T-NF-κB (Cell Signaling, Cat. # 4764), TNF-α (Abcam, Cat. # ab1793), HDAC1 (Cell signaling, Cat. # 5356), HDAC2 (Cell Signaling, Cat. # 5113), HDAC3 (BD Biosciences, Cat. # 611124), arginase 1 (GeneTex; Cat. # GTX109242), arginase 2 (Santa Cruz Biotechnology, Cat. # Sc-20151), tubulin (Sigma, Cat. # T9026), and β-actin (Sigma, Cat. # A1978-200UL). Secondary antibodies (GE Healthcare) were diluted 1:2000 in 5% milk.
7
Flow Cytometry Analysis of Liver Immune Cells
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Liver mononucleated cells were isolated from the livers of naive and MCD-fed mice and purified on a density gradient (Lympholyte®-M, Cedarlane Laboratories Ltd. Burlington, Canada) as described in Crispe (1997) . Cells were then washed with Hank's medium and incubated 30 min with de-complemented mouse serum to block unspecific immunoglobulin binding. The cells were then stained with fluorochrome-conjugated antibodies for CD45, CD11b, Ly6C, MHCII, (eBiosciences, San Diego, CA, USA), F4/80 (Invitrogen, Abingdon, UK) and analyzed with a FACScalibur (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer. Intracellular staining for TNF-α, IL-12 and IL-10 was performed using specific fluorochrome-conjugated antibodies supplied by (eBiosciences, San Diego, CA, USA). AnxA1-and arginase-1-producing cells were detected using polyclonal rabbit antisera against, respectively AnxA1 (Millipore, Temecula, CA, USA) and arginase-1 (Genetex, San Antonio, TX, USA) in combination with phycoerythrin-conjugated antirabbit IgG (Sigma-Aldrich, Milan, Italy).
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