Cells were harvested and lysed in radio immunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors (beyotime Biotechnology). About 40 μg protein per sample was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Epizyme Biomedical Technology) and transferred to nitrocellulose membranes (PALL). The membranes were incubated with 3% bovine serum albumin for 2 h at room temperature (RT) and then incubated overnight at 4 °C with the following primary antibodies: anti-ENO1 (cell signaling technology, 3810), anti-β-Catenin (Cell Signaling Technology, 8480), anti-caspase3 (cell signaling technology, 14220S), anti-p21 (cell signaling technology, 2947S), anti-cyclin D2 (cell signaling technology, 3741S) and anti-GAPDH (cell signaling technology, 5174). Subsequently, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (anti-rabbit IgG, Cell Signaling Technology, 7074S) for 1 h at RT. The specific proteins were detected using enhanced chemiluminescence reagent (Millipore), and the band intensity quantified using quantity one software.
Anti eno1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-ENO1 is a primary antibody developed by Cell Signaling Technology to detect the expression of ENO1 (Enolase 1), a glycolytic enzyme involved in energy production within cells. This antibody can be used in various immunoassay techniques to study the expression and localization of ENO1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Anti cyclin d2, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti pkm2, by Cell Signaling Technology (1 mentions)
Anti ldhα, by Cell Signaling Technology (1 mentions)
Anti gpi, by Cell Signaling Technology (1 mentions)
Anti glut1, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Anti aldoa, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti anxa1, by Cell Signaling Technology (1 mentions)
Anti tet1, by Novus Biologicals (1 mentions)
4 protocols using anti eno1
1
Western Blot Analysis of Protein Markers
2
Metabolic Regulation in T Cell Subsets
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CD4+ Tregs or Teffs after various treatments were washed and harvested. Whole-cell lysates of the T cells were prepared for western blot analyses. Western blots were developed with Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The rabbit polyclonal antibodies used in western blotting are anti-Glut1, anti-LDHα, anti-PKM2, anti-GPI, anti-Eno1, anti-phostho-mTOR, anti- phostho-p70S6K, anti- phostho-4E-BP1, anti-β-actin and anti-GAPDH (Cell Signaling Technology, Boston, USA).
3
Immunoblotting of Cell Lysates
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For immunoblotting experiments, protein samples from the total cell lysates were subjected to SDS-PAGE on 10% or 12% acrylamide/bisacrylamide gels. The protein profiles were electroblotted to PVDF (polyvinylidene difluoride) membranes (Merck Millipore, Burlington, MA, USA) at room temperature for two hours. The antibody anti-ALDOA (Cell Signaling Technologies, Danvers, MA, USA), anti-GAPDH (Cell Signaling Technologies, Danvers, MA, USA), anti-ENO1 (Cell Signaling Technologies, Danvers, MA, USA), and anti-FH (Cell Signaling Technologies, Danvers, MA, USA) incubation procedures and blocking buffer composition were conducted as per the supplier’s instructions.
4
Exosome Protein Profile Analysis
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The cells were collected, washed twice with PBS, and lysed in RIPA buffer containing protease inhibitors. After determining the protein concentration with a BCA Protein Assay Reagent Kit (Pierce), equal amounts of protein were separated on 8% SDS-PAGE, electrically transferred to PVDF membrane, and blocked with 5% skim milk. The membranes were incubated with anti-CD63 (1:500; Abcam, China), anti-TSG101 (1:800; Abcam), anti-CD81 (1:1000; Abcam), anti-CD9 (1:1000; Cell Signaling Technology, China), anti-ENO1 (1:800; Cell Signaling Technology), anti-KRT19 (1:800; Cell Signaling Technology), anti-ANXA1 (1:800; Cell Signaling Technology), anti-PTEN (1:800; Cell Signaling Technology, China), anti-TET1 (1:500; Novus Biologicals, China), anti-TET2 (1:500; Cell Signaling Technology), anti-TET3 (1:500; Novus Biologicals), anti-Akt (1:1000; Cell Signaling Technology), anti-p-Akt (1:1000; Cell Signaling Technology) or anti-beta-actin (1:1000; Cell Signaling Technology) primary antibody overnight at 4 °C. After washing, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Cell Signaling Technology) at room temperature for 1 h. Finally, the membranes were incubated with West Femto chemiluminescence substrate (Pierce), and images were visualized and recorded.
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