Protein lysates (20 to 40 µg) from Mo-DC were subjected to western blot analysis. Membranes were probed with primary antibodies: anti-pIRF3 at Ser 396 (EMD Millipore), anti-IRF3 (IBL, Japan), anti-IRF7 (EMD Millipore), anti-RIG-I (EMD Millipore), anti-IFIT1 (Thermo Fisher Scientic), anti pSTAT1 at Tyr701 (Cell Signaling), anti-STAT1 (Cell Signaling), anti–pIκBα at Ser32 (Cell Signaling), anti-IκBα (Cell Signaling), anti-p47phox at Ser359 (AssayBioTech), anti-p47phox (Sigma Aldrich), anti-STING (Cell Signaling), anti-gp91phox (Santacruz Biotechnology), anti-Nrf2 (Cell Signaling), anti-β-actin (Odyssey, USA). Antibody signals were detected by immunofluorescence using the IRDye 800CW and IRDye 680RD secondary antibodies (Odyssey, USA) and the LI-COR imager (Odyssey, USA). Protein expression levels were determined and normalized to β-actin using the ImageJ software (National Institutes of Health, Bethesda, USA).
Anti gp91phox
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-gp91phox is a primary antibody that recognizes the gp91phox subunit of the NADPH oxidase complex. gp91phox is an essential component of the NADPH oxidase enzyme, which plays a crucial role in the generation of reactive oxygen species in phagocytic cells. This antibody can be used for the detection and analysis of gp91phox expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti p47phox, by Santa Cruz Biotechnology (4 mentions)
Anti p47phox, by Merck Group (2 mentions)
Anti p22phox, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Anti sting, by Cell Signaling Technology (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
Anti irf3, by Tecan (1 mentions)
Anti rig 1, by Merck Group (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Bio-Rad (1 mentions)
Mnsod, by Enzo Life Sciences (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Ted Pella (1 mentions)
Alexa fluor, by Thermo Fisher Scientific (1 mentions)
Avertin, by Merck Group (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
P47phox, by Santa Cruz Biotechnology (1 mentions)
9 protocols using anti gp91phox
1
Comprehensive Immune Signaling Profiling
2
Protein Expression Analysis of Artery Samples
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Stored artery segments were homogenized in lysis buffer containing 50 mM glycerophosphate, 100 µM sodium orthovanadate, 2 mM magnesium chloride, 1 mM EGTA, 0.5% Triton X-100, 1 mM DL-dithiothreitol, 20 µM pepstatin, 20 µM leupeptin, 0.1 U/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride and then incubated on ice for 1 hr. The sample was centrifuged at 1,000 g for 15 min at 1°C, and the supernatant was collected. The total protein was measured by a BCA kit (Bio-Rad). To examine each protein expression, equal amounts of protein (50 µg) from each sample were loaded on each lane and electrophoresed in 4–20% Tris-Glycine gel (Invitrogen) and then transferred onto a polyvinylidene difluoride membrane (Millipore). After being incubated for 2 hrs in 6% dried milk in TBS-Tween buffer, the membrane was incubated overnight at 4°C with specific primary antibody in blocking buffer (anti-p47phox and anti-gp91phox, Santa Cruz Biotech at 1∶200 dilution, MnSOD 1∶1000 dilution, Enzo; eNOS 1∶300 dilution, BD). The membrane was then rinsed and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 hrs at 1∶5,000 dilutions in blocking buffer. All samples from each group were also probed with anti-GAPDH antibody (1∶5000, Abcam) to correct for sample loading.
3
Multimodal Brain Immunolabeling Protocol
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Animals were deeply anesthetized using 0.016 mL/g body weight of a 2.5% solution of avertin (2,2,2 tribromoethanol, Sigma-Aldrich, St. Louis, MO) in 0.9% saline (Sigma-Aldrich, St. Louis, MO) and transcardially perfused with 4% paraformaldehyde (Ted Pella, Redding, CA). Following perfusion, brains were removed and immersed in 0.1 M PBS containing 30% sucrose for 48 h, frozen in OCT compound (Sakura, Netherlands), and serially sectioned into 40-μm-thick coronal sections stored at −80 °C and immunolabeled as previously described [17 (link)] using the following antibodies: mouse monoclonal anti-ankyrinG (ankG; NeuroMab, Davis, CA; N106/36; 1:500), rabbit polyclonal anti-Iba-1 (Wako Chemicals, Richmond, VA; 019-19741; 1:1,000), mouse monoclonal anti-NeuN (Millipore, Billerica, MA; MAB377; 1:1000), mouse monoclonal anti-gp91-phox (Santa Cruz, Dallas, TX; sc-130543; 1:500), mouse monoclonal anti-NaV1.6 (NaV1.6; NeuroMab, Davis, CA; K87A/10; 1:200). All secondary antibodies were obtained from Invitrogen Life Technologies (Grand Island, NY; Alexa™ Fluor) and used at a dilution of 1:500.
4
Western Blot Analysis of Cellular Proteins
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Lysates for Western blot analysis were prepared from cells as follows. Total cell protein was determined using bicinchoninic acid (BCA) reagents following the manufacturer’s instructions. Standard SDS-PAGE was performed and proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane and detected with either polyclonal anti-gp91phox (Santa Cruz, SC130543) or p47phox (Santa cruz, SC17845) antibodies. β-actin was used as a loading control using an anti-β-actin antibody. Goat-anti mouse IgG (Santa Cruz, SC-2005) antibody was used as a secondary antibody conjugated with horseradish peroxidase. Bands were visualized by enhanced chemiluminescence detection using Clarity reagent (Biorad).
5
Western Blot Analysis of Cardiac Protein Markers
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Cells or mice heart tissues were lysed using RIPA buffer containing a 1:100 dilution of protease inhibitor and phosphatase inhibitor (Sigma). Protein concentrations were determined by a BCA protein assay (Pierce), and equal protein samples were separated by SDS-PAGE. Proteins were transferred electrophoretically to polyvinylidene difluoride membranes (Millipore), and then incubated in Tris-buffered saline containing 0.1 % Tween 20 (TBST) with 5 % milk for 1 h at room temperature. Membranes were then incubated with primary antibodies as follows: anti-p66shc (1:1000, Santa Cruz), anti-p47phox (1:1000, Santa Cruz), anti-gp91phox (1:1000, Santa Cruz), anti-α-SMA (1:1000, Abcam), anti-ERK (1:1000, Santa Cruz), anti-pERK (1:1000, Santa Cruz), anti-collagen I (1:500, Bioworld), anti-periostin (1:500, Santa Cruz) and anti-TGF-β (1:500, Santa Cruz). Anti-β-actin antibody (1:2000, Santa Cruz) was used as the internal control. After four washes in TBST, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies. The washes were repeated, and the membranes were then treated with Super Signal Substrate Western Blotting Reagent (Millipore). The bands were quantified using BioRad Quantity One imaging software.
6
Oxidative Stress Signaling Pathway
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Diphenyleneiodonium chloride (DPI), Gö6976, U0126, SB202190, SP600125, and helenalin were obtained from Biomol (Plymouth Meeting, PA). Apocynin was from ChromaDex (Santa Ana, CA). N-acetylcysteine (NAC), tricarbonyldichlororuthenium (II) dimer (CORM-2) and ruthenium (III) chloride (inactive CORM-2) were purchased from Sigma (St. Louis, MO). Anti-GAPDH antibody was obtained from Biogenesis (Boumemouth, UK). Anti-p47phox, anti-HO-1, anti-Gsα, anti-gp91phox, anti-β-actin, and anti-cPLA2 antibodies were from Santa Cruz (Santa Cruz, CA). Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-PKCα/βII, anti-phospho-p65, and anti-phospho-IKKα/β antibodies were from Cell Signaling (Danvers, MA). Dihydroethidium (DHE) was from Molecular Probes (Eugene, OR).
7
Hippocampal and Hypothalamic Protein Analysis
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Total protein was prepared by homogenising hippocampal and post-hypothalamic tissues in lysis buffer containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail. The sample was subsequently incubated for 1 h at 4 °C. The protein extracts (20 μg/sample based on the Bicinchoninic Acid (BCA) protein assay, Pierce Chemical Co., Rockford, IL, USA) were resolved on a 6% polyacrylamide gel and transferred to a PVDF membrane (GE Healthcare, Buckinghamshire, UK). The membranes were incubated in the appropriate anti-P-TauT231 (ab151559), anti-P-AktS473 (4060, Cell Signaling Technology, Beverly, MA, USA), anti-P-GSK-3bS9 (05-643, EMD Millipore, Billerica, MA, USA), anti-P-GSK-3βY216 (ab75745) antibodies at 1:1000 in PBST with 5% BSA at 4 °C overnight, anti-amyloid precursor protein (ab12266), anti-gp91-phox (sc5827, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Tau (ab80579), anti-Akt (9272), anti-p67-phox (sc7663), anti-GSK-3β (07-389), anti-p47-phox (sc14015), anti-p22-phox (sc11712), or anti-caspase 3 (9662) antibodies at 1:1000 in PBST with 5% BSA at room temperature (RT) for 1 h. The membranes were then incubated in an HRP-labelled goat anti-rabbit secondary antibody at 1:10,000. The membranes were developed using an ECL-Plus detection kit (GE Healthcare).
8
Immunoblotting of Phagocyte Proteins
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Proteins were separated by electrophoresis in polyacrylamide gel (8 or 15% gels), transferred to PVDF membranes (Bio-Rad), and immunoblotted. The primary antibodies used were: anti-p47phox (SAB 4502810, Sigma Aldrich), anti-gp91phox (Sc-74514, Santa Cruz), anti-GAPDH (G9545, Sigma Aldrich), anti-CD81 (555675, BD Biosciences), anti-CD63 (Sc-15363, Santa Cruz), anti-caspase-1 (Sc-56036, Santa Cruz), anti-NLRP3 (MA5-32255, Thermo Fisher), anti-ASC (Sc-271054, Santa Cruz). Antigen-antibody reactions were detected by ECL (Amersham Biosciences), and blots were quantified using ImageJ Lab software. Results were normalized to GADPH.
9
Western Blot Analysis of Oxidative Stress Markers in Brain
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Western blot analysis using the rostral side of each brain was performed according to our previous method.29 Primary antibodies used were as follows: anti‐gp91phox (91 kDa) (×2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti‐p22phox (22 kDa) (×1000, Santa Cruz Biotechnology, Inc.), anti‐p67phox (67 kDa) (×5000, BD Biosciences, San Jose, CA), anti‐occludin (65 kDa) (×30 000, Invitrogen, Carlsbad, CA), anti‐brain‐derived neurotrophic factor (BDNF) (14 kDa) (×2000, Santa Cruz Biotechnology, Inc.), anti‐ glyceraldehyde‐3‐phosphate dehydrogenase (37 kDa) (×5000, Santa Cruz Biotechnology, Inc.), anti‐cyclooxygenase‐2 (COX‐2) (72 kDa) (×5000; Acris, Herford, Germany), anti‐extracellular‐superoxide dismutase (EC‐SOD) (45 kDa) (×5000, Upstate, Sharlottesville, VA), anti‐copper‐zinc superoxide dismutase (Cu/Zn‐SOD) (19 kDa) (×5000; Stressgen Bioreagents, Victoria, Canada), anti‐manganese superoxide dismutase (Mn‐SOD) (25 kDa) (×5000; Assay Designs, Farmingdale, NY), and anti‐catalase (60 kDa) (×5000; Cell Signaling Technology, Danvers, MA). Intensity of the bands was quantified by using analysis software (Image J; National Institute of Health, Bethesda, MD). In individual samples, each value was corrected for glyceraldehyde‐3‐phosphate dehydrogenase.
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