Equal amounts of proteins (40 μg total protein or 80 μg nuclear protein per lane) were loaded onto a 12.5% gradient 2-amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride (Tris–HCl) sodium dodecyl sulfate–polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% nonfat milk in 1 × TBS, followed by incubation with primary antibodies against total Akt (rabbit monoclonal 1 :1000; Cell Signaling Technology, Danvers, MA), p-Akt (Ser473, rabbit monoclonal 1:2,000; Cell Signaling Technology), total eNOS (rabbit monoclonal 1:1000; 1:250 dilution, Cell Signaling, USA), and p-eNOS (Ser1179, 1:250 dilution, Invitrogen, USA) overnight at 4 °C. After washing with TBST three times, membranes were incubated with horseradish peroxidase-conjugated rabbit or goat secondary antibody (1:10,000 dilution; Kang Chen Biotechnology, Guangzhou, China) for 1 h at room temperature, followed by three washes for 10 min each. Blots were developed using enhanced chemiluminescent reagents (Thermo Fisher Scientific, Pittsburgh, PA, USA) and target band density was scanned using an LAS-3000 detection system. Image J software was used to analyze band intensities.
P enos
Manufactured by Thermo Fisher Scientific
Sourced in United States
The P-eNOS is a laboratory instrument designed for the detection and quantification of phosphorylated endothelial nitric oxide synthase (p-eNOS) in biological samples. It is a research-grade tool used to study cellular signaling and protein regulation in various scientific fields.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Total enos, by Cell Signaling Technology (1 mentions)
Protease inhibitor tablet, by Thermo Fisher Scientific (1 mentions)
Ecl solution, by Cytiva (1 mentions)
Cell lysis buffer, by Applygen (1 mentions)
Beta actin, by EarthOx (1 mentions)
Gapdh, by Merck Group (1 mentions)
Glut4, by Thermo Fisher Scientific (1 mentions)
P irs1, by Cell Signaling Technology (1 mentions)
P akt, by Abcam (1 mentions)
Beclin 1, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Irs 1, by Abcam (1 mentions)
P jak2, by Thermo Fisher Scientific (1 mentions)
Anti lc3, by Merck Group (1 mentions)
P pi3k, by Abcam (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Alexafluor 488 labeled goat anti rabbit, by Wuhan Servicebio Technology (1 mentions)
Ab179463, by Abcam (1 mentions)
5 protocols using p enos
1
Western Blot Analysis of Akt and eNOS
2
Western Blot Analysis of EPC Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein of EPCs were extracted with cell lysis buffer (Applygen Technologies Company, Beijing) supplemented with protease inhibitor tablet (Thermo Scientific). Protein lysates were electrophoresed through SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies against beta actin (1 : 1000, EarthOx, San Francisco, CA, USA), PI3 kinase p110a (1 : 1000, CST, USA), Akt (1 : 5000, CST, USA), p-Akt (1 : 500, CST, USA), and p-eNOS (1 : 1000, Invitrogen, USA). Blots were developed with ECL solution (Amersham, Sweden).
3
Western Blot Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously described8 (link). In brief, total protein samples extracted from untreated and treated tissues or cells were prepared using RIPA buffer containing a protease inhibitor cocktail (Roche, Branford, CT, USA) and a phosphatase inhibitor and were subjected to SDS PAGE (90μg protein per sample). The membranes were then incubated with the following primary antibodies: p-IRS-1 (Cell Signaling Inc. (CST), Danvers, MA, USA,1:800), eNOS (CST,1:1,000), p-eNOS (Thermo Fisher,1:800), IRS-1 (CST,1:1,000), p-AKT (CST,1:1000), AKT (CST,1:1000), p-PI3K (Abcam, Cambridge, MA, USA,1:500), PI3K(Abcam1:1000),GAPDH (CST, 1:1,500), anti-LC3 (1:200, Sigma-Aldrich, St. Louis, MO, USA), Beclin1 (1:200, Sigma-Aldrich), GLUT4 (1:3,000, Thermo Fisher), p-JAK2 and JAK2(CST,1:1000).
4
Immunofluorescence Imaging of Endothelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was performed according to the manufacturer’s protocols. Primary antibodies and corresponding dilutions were as follows: NF-200 (1:200, CST, 2836S), S100β (1:200, Abcam, ab52642), CD31 (1:200, Abcam, ab182981), CD34 (1:200, Abcam, ab81289), VEGFR (1:200, Abcam, ab2349), AKT (1:100, Abcam, ab179463), p-AKT (1:100, Abcam, ab192623), eNOS (1:100, ThermoFisher, PA1-037), p-eNOS (1:200, ThermoFisher, PA1-037). Secondary antibodies and corresponding dilutions were as follows: FITC-labeled goat anti-rabbit (1:200, Goodbio, GB22303), CY3-labeled goat anti-mouse (1:300, Goodbio, GB21301), CY3-labeled goat anti-rabbit (1:300, Goodbio, GB21303), AlexaFluor®488-labeled goat anti-rabbit (1:400, Servicebio, GB25303). Nuclei were labeled with 4′6-Diamidino-2-phenylindole-dihydrochloride (DAPI, Servicebio, G1012). Fluorescence images were acquired with fluorescence microscope (Olympus, Japan). Fluorescence intensity was analyzed with ImageJ software.
5
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The western blot analysis was performed using a previously reported protocol (Guo et al., 2020 (link)). Cells or tissues were disrupted in a RIPA buffer containing protease and phosphatase inhibitors. After protein quantification with a bicinchoninic acid (BCA) kit, proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were electroblotted onto polyvinylidene fluoride (PVDF) membranes. Non-specific binding was blocked using 5% BSA for 1 h at room temperature. Then the membranes were incubated with primary antibodies at 4°C overnight and probed with horseradish peroxidase-conjugated secondary antibodies for 90 min at room temperature. Enhanced chemiluminescence was used to amplify the protein signals. The density of immunoreactive proteins was assessed by using the ImageJ software. The following primary antibodies were used: PLCγ1 (Cell Signaling Technology; 5,690; 1:1000), p-PLCγ1 (Tyr783; Cell Signaling Technology; 14,008; 1:1000), eNOS (Cell Signaling Technology; 32,027; 1:1000), p-eNOS (Ser1179; ThermoFisher Scientific; PA5-105824; 1:1000), AKT (Cell Signaling Technology; 9,272; 1:1000), p-AKT (Ser473; Cell Signaling Technology; 4,060; 1:2000), GAPDH (Affinity; AF7021; 1:5,000).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!