Anti-GAPDH antibody was purchased from Fitzgerald Antibodies (Acton, MA) and was used at a dilution of 1:1,000. Anti-phospho-Akt (Ser473), anti-Akt, and anti-phospho-eNOS (Ser1177) antibodies were purchased from Cell Signaling (Danvers, MA) and were all used at a dilution of 1:1,000. Anti-eNOS antibody (1:500 dilution), anti-mouse IgG (1:10,000 dilution), and anti-rabbit IgG (1:2,000 dilution) antibodies were purchased from Abcam (Cambridge, MA). MK2206 and GDC0068 were purchased from SelleckChem (Houston, TX). Gallein, LY294002, L-NAME, and ODQ were purchased from Tocris (Ellisville, MO). All other chemicals were purchased from Sigma (St. Louis, MO).
Anti phospho enos ser1177
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-eNOS (Ser1177) is a laboratory reagent used to detect the phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1177. This antibody can be used in various immunoassay techniques to measure the levels of phosphorylated eNOS, which is an important regulatory mechanism in the production of nitric oxide.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (4 mentions)
Anti enos, by Cell Signaling Technology (4 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (3 mentions)
Anti enos, by BD (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti cox 2, by Cayman Chemical (2 mentions)
Phosphate buffered saline (pbs), by Welgene (2 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (2 mentions)
Anti phospho enos thr495, by Cell Signaling Technology (2 mentions)
Acetylcholine, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Gallein, by Bio-Techne (1 mentions)
Anti enos antibody, by Abcam (1 mentions)
Anti rabbit igg, by Abcam (1 mentions)
Anti mouse igg, by Abcam (1 mentions)
L name, by Bio-Techne (1 mentions)
Ly294002, by Bio-Techne (1 mentions)
Gdc 0068, by Selleck Chemicals (1 mentions)
18 protocols using anti phospho enos ser1177
1
Antibody-Based Western Blot Analysis
2
Analyzing LOX-1, Akt, and eNOS in HAECs
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HAECs in EGM-2 medium were treated with L1 (50 μg/ml) or L5 (50 μg/ml) from HD patients or phosphate-buffered saline (PBS) and were cultured for 24 hours. Cellular proteins were extracted and subjected to SDS–polyacrylamide gel electrophoresis.23 (link) Immunoblotting was performed with anti-LOX-1 (GeneTex, Inc., Irvine, CA), anti-Akt, anti-phospho-Akt, anti-eNOS, anti-phospho-eNOS (Ser1177), and anti-actin antibodies (Cell Signaling Technology, Danvers, MA). LOX-1 levels were normalized to those of β-actin, phospho-Akt levels were normalized to those of Akt, and phospho-eNOS levels were normalized to those of eNOS.
3
Omentin Attenuates LPS-Induced Vascular Injury
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LPS (E. coli LPS serotype 0111:B4), sodium pentobarbital, Evans Blue dye, collagenase and trypsin were purchased from Sigma (St. Louis, MO, USA). rh-omentin protein was purchased from GeneTex (Irvine, CA, USA). The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-Scr, anti-phospho-Scr (Try416), anti-Akt, anti-phospho-Akt (Ser473), anti-eNOS, anti-phospho-eNOS (Ser1177), anti-NF-κB Rel and anti-phospho-NF-κB Rel (Ser536). Anti-VE-cadherin, anti-pan-cadherin and anti-cleaved caspase-3 antibodies were purchased from Abcam (Cambridge, UK). Anti-GAPDH, anti-CD31, anti-caspase-3, anti-GSK-3β and anti-phospho-GSK-3β (Ser9) antibodies were purchased from Bioworld Technology (Nanjing, China). Ad-β-gal and full-length human omentin (Ad-omentin) were constructed by Genechem (Shanghai, China). Ad-β-gal was used as a control.
4
Protein Expression Analysis by Western Blot
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For the determination of protein expression, cells either untreated or incubated for 30 min with 1 and 100 nM dDAVP were lysed in LDS sample buffer (Thermo Fisher Scientific) and Western Blot analysis was performed as described [13 (link)]. Briefly, 20 μg of proteins were separated on Bolt™ 4–12% Bis-Tris mini protein gel (Thermo Fisher Scientific) and electrophoretically transferred to PVDF membranes (Immobilione-P membrane, Merck). Membranes were incubated for 1 h at RT in TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl; 0.1% Tween) containing 5% non-fat dried milk, then incubated overnight at 4 °C in TBST added with 5% BSA and anti-AVPR1A (Thermo Fisher Scientific), anti-AVPR2 (MyBiosource, distributed by Aurogene S.r.l, Roma, Italy) anti-phospho-eNOS (Ser1177) (Cell Signaling Technology, distributed by Euroclone, Pero, Italy) purified rabbit polyclonal antibodies (1:2000). Actin, detected with a monoclonal antibody (1:2000; Merck, Milano, Italy), was employed as internal standard. Immunoreactivity was visualized with SuperSignal™ West Pico Plus Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with iBright Analysis Software.
5
Immunoblotting for COX-2, eNOS, and Akt
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Immunoblotting using anti-COX-2 (Cayman Chemicals, #160126) was performed on skin harvested from each group. Pharmacological drugs efficiency was confirmed by immunoblotting using anti-phospho-eNOS-Ser1177, anti-phospho-Akt-Ser473 (Cell signalling Technology; Danvers, MA) on treated skin harvested at the maximum of PIV.
6
Cavernosal Protein Expression Analysis
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As described above, the cavernosal tissue was obtained immediately after we performed the cavernosometery. The same quantity of protein extracts (20–50 µg) were separated in sodium dodecyl sulfate polyacrylamide gels, which were then transferred to nitrocellulose membranes. An overnight incubation was performed with primary antibodies, including anti-neuronal nitric oxide synthase (1:2,000; Cell Signaling Technology, Boston, MA, USA), anti-endothelial nitric oxide synthase (eNOS) (1:3,000; BD Biosciences, San Jose, CA, USA), anti-phospho-eNOS (Ser1177, 1:1,000; Cell Signaling Technology), anti-myosin phosphatase target subunit 1 (MYPT1) (1:2,000; Cell Signaling Technology), anti-phospho-MYPT1 (Thr696, 1:1,000; Millipore, Charlottesville, VA, USA), and platelet endothelial cell adhesion molecule-1 (PECAM-1) (1:1,000; Santa Cruz Biotechnology) [3 (link)4 (link)6 (link)7 (link)13 (link)16 (link)]. The bands were visualized using the electrochemiluminescence Western blotting development system. To adjust for any loading differences, the membranes were re-probed with an antibody against monoclonal anti-actin antibodies. The results were then quantified by means of densitometry using Image J analysis software (National Institute of Health).
7
Molecular Mechanisms in HepG2 Cell Culture
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HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). LSECs and endothelial cell medium were obtained from ScienCell Research Laboratory (Carlsbad, CA, USA). Phosphate-buffered saline, penicillin/streptomycin, and trypsin-ethylenediaminetetraacetic acid solutions were purchased from Welgene (Daegu, Korea). SP, phenylmethylsulfonyl fluoride (PMSF), and CDCA were provided by Sigma Aldrich (St. Louis, MO, USA). Anti-GAPDH, anti-ICAM-1 (EP1442Y), and anti-caspase-3 (E87) antibodies (Abcam, Cambridge, MA, USA) were also used. Anti-epithelial cadherin (E-cadherin) (24E10), anti-cleaved caspase-3 (Asp175), anti-endothelial nitric oxide synthase (eNOS), and anti-phospho-eNOS (Ser1177) antibodies and 10X cell lysis buffer were obtained from Cell Signaling Technology (Danvers, MA, USA).
8
Endothelium-Dependent Nitric Oxide Signaling
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All chemicals were commercially available and had the highest purity. DMT was obtained from Orion Pharma (Turku, Finland). L-NAME, MβCD, PP2, rauwolscine, phenylephrine, and acetylcholine were obtained from Sigma Aldrich (St. Louis, MO, USA). Intralipid (20%), which is composed of linoleic acid (53%), oleic acid (24%), palmitic acid (11%), alpha-linolenic acid (8%), and stearic acid (4%), was obtained from Fresenius Kabi AB (Uppsala, Sweden). Anti-eNOS antibody (#610297) was obtained from BD Bioscience (Franklin, NJ, USA). Anti-phospho-eNOS (Ser1177) (#9571), anti-phospho-eNOS (Thr495) (#9574), anti-Src kinase (#2108), anti-phospho-Src kinase (Tyr416) (#2101), anti-caveolin-1 (#3238), and anti-phospho-caveolin-1 (Tyr14) (#3251) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). All chemical concentrations were expressed as the final molar concentration.
9
Western Blot Analysis of Endothelial Proteins
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Protein samples collected from mBMECs homogenates were electrophoresed through a 10% SDS-polyacrylamide gel and transferred onto an Immun-Blot® PVDF membrane (Bio-Rad; cat# 1620177). Non-specific binding was blocked by 3% BSA in 0.1% Tween-20 TBS for 1 hour. The blots were then incubated overnight at 4°C with primary antibodies: anti-phospho-eNOS Ser1177 (1:1,000, Cell Signaling Technology, CST, Danvers, MA, USA; cat# 9570S), anti-eNOS (1:1,000, CST; cat# 32027S), anti-phospho-Akt Ser473 (1:1,000, CST; cat# 9271S), anti-Akt (1:1,000, CST; cat# 9272S), MCP1 (1:1,000, CST; cat# 2027), GAPDH (1:5000, CST; cat# M00227). The blots were eventually incubated with goat anti-rabbit IgG (H + L) horseradish peroxidase conjugate (1:10,000, Invitrogen; cat# G21234) prior to chemiluminescence detection by the ChemiDoc MP Imaging System (Bio-Rad) with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
10
Endothelial Cell Characterization Protocol
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All tissue culture reagents were from Euroclone SpA except ECGS and heparin (Sigma Aldrich, cat #E2759 and #H3149, respectively). NG-Nitro-L-arginine methyl ester (L-NAME, cat #N5751), methylcellulose (cat #M0512), FITC-labelled phalloidin (cat #P5282), and DAPI (cat #D9542) were from Sigma Aldrich; collagenase (cat #17454) and rat tail collagen I (cat #47254) from Serva; recombinant human VEGF165 (cat #100) from Peprotech; mitomycin (cat #BIA-M1183) from Tebu-bio. Primary antibodies used were: mouse monoclonals anti-eNOS (BD Transduction Laboratories, cat #610296) and anti-β-actin (Sigma Chemicals, cat #A2228), and rabbit polyclonal anti phospho-eNOS (Ser1177) (Cell Signalling Technology, cat #9571). HRP-conjugated secondary antibodies were from Dako (cat #P0260 and #P0399 for rabbit anti-mouse and swine anti-rabbit antibodies, respectively). A goat anti-mouse CY3 (cat #29-0382-75, GE Healthcare) was used in immunofluorescence experiments to detect eNOS.
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