Liver samples were processed and western blot performed as described42 (link). Used primary antibodies: KLF2 (Santa Cruz Biotech, Santa Cruz, CA), phosphorylated eNOS at Ser1177 (Cell Signaling, Danvers, MA), and total eNOS (BD Transduction Laboratories, Lexington, KY), all 1:1000. Blots were revealed by chemiluminescence and protein expression was determined by densitometric analysis using the Science Lab 2001, Image Gauge (Fuji Photo Film, Düsseldorf, Germany). Blots were also assayed for β-actin (Sigma-Aldrich) content as standardization of sample loading.
Total enos
Manufactured by BD
Sourced in United States
Total eNOS is a lab equipment product that measures the activity of the endothelial nitric oxide synthase (eNOS) enzyme. eNOS is responsible for the production of nitric oxide, a critical signaling molecule in the cardiovascular system. The Total eNOS product provides a quantitative assessment of eNOS activity, which can be used in research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Phospho enos ser1177, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosphorylated enos at ser1177, by Cell Signaling Technology (1 mentions)
Science lab 2001 image gauge, by Fujifilm (1 mentions)
Collagen 1α1, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Image studio lite, by LI COR (1 mentions)
3 nitrotyrosine, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Dynasore, by Cayman Chemical (1 mentions)
Cytochalasin d, by Cayman Chemical (1 mentions)
P enos, by Cell Signaling Technology (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Vybrant did cell labeling dye, by Thermo Fisher Scientific (1 mentions)
Grp94, by Cell Signaling Technology (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Tsg101, by Santa Cruz Biotechnology (1 mentions)
8 protocols using total enos
1
Western Blot Analysis of Liver Samples
2
Liver Protein Expression Analysis
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Liver samples were processed, and Western blot performed as described [25 (link)]. The primary antibodies used were 3-Nitrotyrosine (N5538, Sigma Aldrich, San Luis, MO, USA), cleaved caspase-3 (9661, Cell Signalling, Danvers, MA, USA), alpha smooth muscle actin (α-SMA) (A2547, Sigma Aldrich, San Luis, MO, USA), collagen 1α1 (84336, Cell Signalling, Danvers, MA, USA), cellular retinol binding protein (CRBP1) (sc-271208, Santa Cruz), phosphorylated moesin at Thr558 (sc-12895, Santa Cruz, CA, USA), total moesin (sc-13122, Santa Cruz, CA, USA), phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 (9571, Cell Signaling, Danvers, MA, USA), and total eNOS (610297, BD Transduction Laboratories, San Jose, CA, USA), all 1:200. Blots were revealed by chemiluminescence and protein expression was determined by densitometric analysis using the Image Studio Lite (LI-COR, Lincoln, USA). Blots were also assayed for α-tubulin (1:1000, Sigma-Aldrich, San Luis, MO, USA) content as standardization of sample loading.
3
Quantifying Phospho-eNOS Protein Levels
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Immunoblotting was performed as previously described [23] (link) with antibodies in the following concentrations: phospho-eNOS Ser1177 1∶1000 (Cell Signaling, Danvers, MA), total eNOS 1∶2500 (BD Transduction Laboratories, Lexington, KY). Densitometry was performed using Image J.
4
Exosome Characterization Using Antibodies
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Various antibodies were purchased from the following suppliers: HIV-1 Tat (#ab43014) from Abcam, Cambridge, MA, USA; against phospho-DRP1 (#3455), Alix (#2171), GRP94 (#2104), and p-eNOS (#9571) from Cell Signaling Technology, Danvers MA, USA; against CD81 (#sc-23962), CD63 (#sc-5275), and TSG101 (#sc-7964) from Santa Cruz Biotechnology, Dallas, TX, USA; β-actin (#A5441) from Sigma-Aldrich; total DRP-1 (#611112), total eNOS (#610296) from BD Transduction Laboratory, San Jose, CA, USA. Vybrant DiD cell-labeling dye for exosome staining was purchased from Molecular Probes (Eugene, OR, USA). Dihydroethidium (DHE) (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect cytosolic superoxide and MitoSOX Red (Molecular Probes) for the detection of mitochondrial superoxide. To label mitochondria, MitoTracker Red and MitoTracker Green were used (Molecular Probes). Phorbol 12-myristate 13-acetate (PMA), PX866, Dynasore, and Cytochalasin D were from Cayman Chemical (Ann Arbor, MI), Ionomycin was from Sigma-Aldrich, and recombinant human IL-2 (rIL-2) was from Miltenyi Biotec (Auburn, CA, USA). GW4896 was from Selleckchem (Houston, TX).
5
Immunohistochemical Analysis of VCCC Filter
6
Protein Expression Analysis in Penis Tissue
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Penis tissue and MCECs were lysed in RIPA buffer (Sigma-Aldrich) Supplemented with protease inhibitors (GenDEPOT, Katy, TX, USA) and phosphatase inhibitors (GenDEPOT). Equal amounts of protein (40 µg/lane) were resolved by SDS-PAGE on 8% to 15% gels, and then transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk for 1 hour at room temperature, membranes were probed with antibodies against Hsp70 (Abcam; 1:1,000), neurotrophin-3 (NT-3; Santa Cruz Biotechnology; 1:1,000), nerve growth factor (NGF; Santa Cruz Biotechnology; 1:1,000), BDNF (Santa Cruz Biotechnology; 1:1,000), pro and cleaved caspase-3 (Novus Biologicals, Centennial, CO, USA; 1:1,000), phosphorylated-eNOS (p-eNOS; Invitrogen; 1:500), total eNOS (BD Biosciences, Franklin Lakes, NJ, USA; 1:500), phosphorylated-Akt (p-Akt; Cell Signaling; 1:1,000), total Akt (Cell Signaling; 1:1,000) p-PI3K (Cell Signaling; 1:1,000), total PI3K (Cell Signaling; 1:1,000) phosphorylatedp-ERK (p-ERK; Cell Signaling; 1:1,000), total ERK (Cell Signaling; 1:1,000), p-NF-κB (Cell Signaling; 1:500), total NF-κB (Cell Signaling; 1:500), Cse (Thermo Fisher Scientific; 1:1,000), SDF1 (Abcam; 1:1,000), HO-1 (Santa Cruz Biotechnology; 1:500), and/or β-actin (Abcam; 1:5,000), as described in the text. The results were quantified densitometrically using Image J software.
7
Immunoblotting for Phospho-eNOS Quantification
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For immunoblotting, we homogenized tissues in RIPA buffer (Wako Pure Chemical Industries, Ltd., Tokyo) containing protease inhibitors (Takara Bio Inc., Shiga, Japan) and a phosphatase inhibitor (Nacalai Tesque, Kyoto, Japan) and collected the supernatants. Proteins (5 μg/lane) were separated on 5–20% gradient SDS–polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (GE Healthcare Bio Sciences, Piscataway, NJ). After blocking with TBS-T buffer containing either 3% bovine serum albumin or 5% skim milk, the membranes were incubated with antibodies against phospho-eNOS (Ser1177, 1:1000, Cell Signaling Technology, Beverly, MA), phospho-eNOS (Thr495, 1:1000, Cell Signaling Technology) and total eNOS (1:1000, BD Biosciences, San Diego, CA) or a peroxidase-conjugated antibody against β-actin (1:50000, Sigma–Aldrich, St. Louis, MO) [29 (link)] and antibody binding was detected with horseradish peroxidase-conjugated secondary antibodies (1:2000; Chemicon) using an enhanced chemiluminescence system (GE Healthcare Japan, Tokyo). The band intensity of phospho-eNOS (Ser1177 and Thr495) was scanned in gray scale at the maximum resolution of at least 600 dpi using NIH Image J 1.47 and arbitrary ratio normalized to the band intensity of total-eNOS were used.
8
Kidney Protein Extraction and Western Blot
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Snap-frozen kidney pieces were homogenized in CellLyticTM MT Cell Lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA). Protein concentration was determined with a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). 35 µg of homogenized protein were prepared/well in 2x Laemmli buffer (Bio-Rad laboratories, Hercules, CA, USA). Tissue lysates were run on a 4–15% Criterion TGX precast gel (Bio-Rad Laboratories). Membranes were incubated with primary antibody overnight at 4 °C as follows: KIM-1 (1:1500 or 0.13 µg/mL, R&D Systems, Minneapolis, MN, USA), NGAL (1:1500 or 0.13 µg/mL, R&D Systems, Minneapolis, MN, USA), total eNOS (1:1000 or 0.25 µg/mL, BD Biosciences, Franklin Lakes, NJ, USA), p1177 eNOS (1:1000 or 0.25 µg/mL, BD Biosciences, Franklin Lakes, NJ). Secondary antibodies were applied the next day for 1 h at room temperature (1:10,000 KPLaboratories, Gaithersburg, MD, USA) and blots were developed using Clarity™ Western Blotting Substrate (Bio-Rad Laboratories). A ChemiDoc™ Touch Imaging System was used for exposure (Bio-Rad Laboratories). Densitometry was performed in Image J software [38 ], and bands of interest were normalized to total protein as determined by a simultaneously run Coomassie stained gel.
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