For in vitro experiments, hepatocytes were washed with cold phosphate-buffered saline (PBS), collected in lysis buffer (Cell Signaling Technology), sonicated, and centrifuged at 16,000g for 15 min, after which the supernatant was collected. For in vivo experiments, snap-frozen liver (median lobe) was homogenized in lysis buffer and centrifuged at 16,000g for 15 min, after which the supernatant was collected. Protein concentrations were determined with the BCA (bicinchoninic acid) protein assay kit (Thermo Fisher Scientific). Loading buffer was added to the samples, which were then resolved by 10 or 15% SDS–polyacrylamide gel electrophoresis. Samples were then transferred onto a polyvinylidene difluoride membrane at 250 mA for 2 hours. The membrane was blocked in 5% milk for 1 hour and then incubated overnight with primary antibody in 1% milk. Membranes were washed in tris-buffered saline containing Tween (TBS-T) for 10 min, incubated with horseradish peroxidase– conjugated secondary antibody for 1 hour, and then washed for 1 hour in TBS-T, before being developed for chemiluminescence (Thermo Fisher Scientific). The primary antibodies used were anti-iNOS (1:1000, R&D Systems Inc.), anti-TNFR1, anti-TACE, and anti-GAPDH (1:1000 for all antibodies, Abcam). Protein band intensities were quantified with ImageJ software (National Institutes of Health).
Anti inos
Manufactured by R&D Systems
Sourced in United States
Anti-iNOS is a lab equipment product that functions as an antibody specific for the inducible nitric oxide synthase (iNOS) protein. iNOS is an enzyme responsible for the production of nitric oxide, which plays a role in various biological processes. The Anti-iNOS product is used for the detection and analysis of iNOS in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Anti tnfr1, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Human ifn γ, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Anti icam 1, by Santa Cruz Biotechnology (1 mentions)
Q vd oph, by Cayman Chemical (1 mentions)
Parthenolide, by Cayman Chemical (1 mentions)
Sb203580, by Cayman Chemical (1 mentions)
Sp600125, by Cayman Chemical (1 mentions)
Anti ho 1, by Enzo Life Sciences (1 mentions)
Ly294002, by Cayman Chemical (1 mentions)
A549 cells, by RIKEN BioResource Center (1 mentions)
A1rmp multiphoton confocal microscope, by Nikon (1 mentions)
Opal 4 color manual ihc kit, by PerkinElmer (1 mentions)
Anti arg1, by Santa Cruz Biotechnology (1 mentions)
Anti cd68, by Abcam (1 mentions)
Anti arginase 1, by R&D Systems (1 mentions)
6 protocols using anti inos
1
Western Blot Analysis of Protein Levels
2
Culturing Human Lung Carcinoma A549 Cells
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Human lung carcinoma type 2 epithelium-like A549 cells, purchased from Riken BioResource Center Cell Bank (Tsukuba, Ibaraki, Japan), were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Life Sciences, MO, USA) with 10% heat-inactivated fetal bovine serum (FBS; GE Healthcare Japanhealth, Tokyo, Japan), 100 U/ml penicillin, 100 mg/ml streptomycin in 10 cm dishes at 37 °C in a humidified atmosphere of 5% CO2. A549 cell line has been characterized as an in vitro model of alveolar type 2 pneumocytes of the human lung because of capacity of secreting lung surfactant-associated glycoproteins [8 (link)].
Reagents were purchased commercially as follows: human IL-1β (Humanzyme, Chicago, IL, USA); human TNF-α (Prospec, Rehovot, Israel), human IFN-γ (Peprotech, NJ, USA); dexamethasone, Fas ligand (super Fas Ligand) (Enzo, PA, USA); RIPA buffer (Thermo Fischer Scientific, Tokyo, Japan); lipopolysaccharide (LPS: E. coli O111:B4, Sigma-Aldrich Life Sciences); rapamycin, parthenolide, SP600125, SB203580, U0123, LY294002, and QVD-OPh (Cayman Chemical, MI, USA).
Antibodies were as follows: anti-iNOS (R&D systems, MN, USA); anti-HO-1 (Enzo); anti-beta actin (MBL, Nagoya, Japan); anti-JNK (Abcam Japan, Tokyo, Japan); anti-cox-2 (BD Japan, Tokyo, Japan); anti-ICAM-1 (Santa Cruz Biotechnology, TX, USA). All of the other antibodies were purchased from CST Japan, Tokyo, Japan.
Reagents were purchased commercially as follows: human IL-1β (Humanzyme, Chicago, IL, USA); human TNF-α (Prospec, Rehovot, Israel), human IFN-γ (Peprotech, NJ, USA); dexamethasone, Fas ligand (super Fas Ligand) (Enzo, PA, USA); RIPA buffer (Thermo Fischer Scientific, Tokyo, Japan); lipopolysaccharide (LPS: E. coli O111:B4, Sigma-Aldrich Life Sciences); rapamycin, parthenolide, SP600125, SB203580, U0123, LY294002, and QVD-OPh (Cayman Chemical, MI, USA).
Antibodies were as follows: anti-iNOS (R&D systems, MN, USA); anti-HO-1 (Enzo); anti-beta actin (MBL, Nagoya, Japan); anti-JNK (Abcam Japan, Tokyo, Japan); anti-cox-2 (BD Japan, Tokyo, Japan); anti-ICAM-1 (Santa Cruz Biotechnology, TX, USA). All of the other antibodies were purchased from CST Japan, Tokyo, Japan.
3
Multicolor IHC Analysis of Bone Marrow
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Formalin‐fixed paraffin‐embedded bone marrows were analyzed using the Opal 4‐Color Manual IHC Kit (catalog no. NEL810001KT, Perkin Elmer) per the manufacturer's instructions. A Nikon A1RMP+ multiphoton confocal microscope was used to capture the fluorescent images. The primary antibodies were anti‐CD68 (catalog no. ab222914, Abcam), anti‐iNOS (catalog no. MAB9502, R&D Systems), and anti‐ARG1 (catalog no. sc166920, Santa Cruz).
4
Western Blot Analysis of Immunomodulatory Proteins
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Freshly isolated spDC, TIDC, or murine normal hepatocytes were lysed at 4°C for 20 min in a RIPA buffer containing protease inhibitors (2.5 μg/mL pepstatin, 10 μg/mL aprotinin, 5 μg/mL leupeptin, and 0.1 mM PMSF). After centrifugation, protein concentration in the supernatant was determined using a Bio-Rad protein assay (Hercules). Thirty micrograms of proteins was separated by SDS-PAGE (12% polyacrylamide gel for IDO and arginase-1 detection and 7% for inducible NOS (iNOS) detection). Proteins were electrotransferred onto a nitrocellulose membrane. The membrane was blocked in Tris Buffered Saline (TBS) with 1% Tween-20 and 5% skim milk and incubated overnight (4°C) with anti-mouse-IDO monoclonal antibody (1 : 5000, Enzo Life Sciences), anti-arginase-1 (1 : 2000, R&D Systems), and anti-iNOS (1 : 500, R&D Systems). The membrane was washed and incubated (room temperature, 2 h) with HRP-conjugated secondary antibody. Immunoblots were then developed using an enhanced chemiluminescence (ECL) reagent kit from Santa Cruz Biotechnology, according to the manufacturer's protocol.
5
Automated Western Blot for iNOS Quantification
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BV-2 cells were seeded in 24-well plates at a density of 1.0 × 105 cells/well and incubated for 24 h. Afterward, the cells were pretreated with ALR-ELNs (20 μg/mL) for 3 h and stimulated with 0.5 μg/mL LPS, after which the cells were incubated for 12 h. Then, the cells were collected and washed with phosphate-buffered saline followed by the addition of RIPA buffer (Santa Cruz Biotechnology, Dallas TX, United States). Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific).
Automated western blots were performed using the Wes Western Blot System (ProteinSimple, San Jose, CA, United States) according to the manufacturer’s protocol and recommendations. Cell lysates were diluted to 0.2 mg/mL, and a size assay was run on a 25-well plate. The assay parameters were as follows: 25 min separation time, 375 V, 5 min antibody diluent, 30 min primary antibody incubation, and 30 min secondary antibody incubation. The following primary antibodies were used: anti-iNOS (dilution 1:25; mouse, R&D Systems) and anti-β-actin (1:250; rabbit, Cell Signaling Technology, Danvers, MA, United States). Densitometric analysis was performed using the Compass software (ProteinSimple) and protein quantification was conducted using the area under the curve calculation method.
Automated western blots were performed using the Wes Western Blot System (ProteinSimple, San Jose, CA, United States) according to the manufacturer’s protocol and recommendations. Cell lysates were diluted to 0.2 mg/mL, and a size assay was run on a 25-well plate. The assay parameters were as follows: 25 min separation time, 375 V, 5 min antibody diluent, 30 min primary antibody incubation, and 30 min secondary antibody incubation. The following primary antibodies were used: anti-iNOS (dilution 1:25; mouse, R&D Systems) and anti-β-actin (1:250; rabbit, Cell Signaling Technology, Danvers, MA, United States). Densitometric analysis was performed using the Compass software (ProteinSimple) and protein quantification was conducted using the area under the curve calculation method.
6
Western Blot Analysis of iNOS and p38 MAPK
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Freshly isolated PMNs were lysed by sonification (SONICS Vibra Cell) in the presence of protease inhibitor cocktail (Sigma-Aldrich). The lysates were suspended in Laemmli buffer (Bio-Rad Laboratories) and electrophoresed (Bio-Rad Laboratories Mini-PROTEAN® Tetra Cell) on sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad). The resolved protein was transferred onto nitrocellulose (Bio-Rad Laboratories Mini-PROTEAN® Tetra Cell). The nitrocellulose was incubated (Milipore SNAP i.d.TM Protein Detection System) with the primary monoclonal anti-iNOS (1:10,000 R&D Systems) or polyclonal antibody antiphospho-p38α MAPK active form (1:10,000 ABR Affinity BioReagents). After washing with D r a f t 8 0.1% TBS-T (Bio-Rad Laboratories) the membrane was incubated with alkaline phosphataselabelled anti-mouse or anti-rabbit IgG antibodies (Vector Laboratories). Immunoreactive protein bands were visualized using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) Liquid Substrate System (Sigma-Aldrich). The density of iNOS and p38 MAP kinase bands was determined using ImageJ software and quantified with arbitrary units.
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