Hematoxylin and eosin (H&E) staining was performed on all paraffin blocks with the tissue samples obtained from both patients and PDXs. The anti-IRS2 (Abcam, Cambridge, MA, USA, EPR904(2)) and anti-phospho-AKT (Cell Signaling Technology, 3787) antibodies were used for IRS2 and p-AKT immunohistochemical staining. The sections (3 μm) were deparaffinized and rehydrated, and antigen retrieval was performed in a citrate buffer (pH 6.1) at 95°C for 40 min. Diaminobenzidine was used as the chromogen. The sections were counterstained with hematoxylin. The BenchMark XT IHC/ISH staining instrument (Ventana Medical Systems, Tucson, AZ, USA) was utilized. IRS2-expressing cancer tissues were used as positive controls.
Anti irs2
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-IRS2 is a primary antibody that recognizes the insulin receptor substrate 2 (IRS2) protein. IRS2 is a signaling adapter protein that plays a key role in insulin and growth factor signaling pathways. This antibody can be used for the detection and analysis of IRS2 in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (2 mentions)
Anti glut4, by Abcam (2 mentions)
Anti irs 1, by Abcam (2 mentions)
Anti insr, by Abcam (2 mentions)
Benchmark xt ihc ish staining instrument, by Roche (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Anti t akt, by Abcam (1 mentions)
Anti p akt t308, by Abcam (1 mentions)
Anti p akt s473, by Abcam (1 mentions)
Anti tfam, by Abcam (1 mentions)
Anti errα, by Abcam (1 mentions)
Anti nrf1, by Abcam (1 mentions)
Anti pgc 1α, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
6 protocols using anti irs2
1
Immunohistochemical Analysis of IRS2 and p-AKT
2
Protein Expression Analysis via Western Blotting
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Total protein was extracted using RIPA buffer supplemented with protease inhibitors. After cell lysis on ice for 30 min, the lysate was centrifuged at 12,000 rpm for 20
min, and the supernatant was stored at –80°C. Before electrophoresis, the samples were heated to 100°C for 5 min. Each sample was separated by 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. After incubation in blocking buffer for 1 h at
37°C, the membrane was incubated overnight at 4°C with anti-pAKT (1:1000, Cell Signaling Technology, Boston, MA, USA), anti-AKT (1:1000, Cell Signaling Technology),
anti-IRS2 (1:500, Abcam, Cambridge, England) or β-actin (1:2000, Abcam) antibodies as a loading control. After washing, the membranes were incubated with secondary
antibody conjugated to horseradish peroxidase at 37°C for 30 min. Finally, the immunoreactive bands were visualized using a Super Signal West Pico Trial Kit (Pierce,
Rockford, Illinois, USA) according to the manufacturer’s instructions.
min, and the supernatant was stored at –80°C. Before electrophoresis, the samples were heated to 100°C for 5 min. Each sample was separated by 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. After incubation in blocking buffer for 1 h at
37°C, the membrane was incubated overnight at 4°C with anti-pAKT (1:1000, Cell Signaling Technology, Boston, MA, USA), anti-AKT (1:1000, Cell Signaling Technology),
anti-IRS2 (1:500, Abcam, Cambridge, England) or β-actin (1:2000, Abcam) antibodies as a loading control. After washing, the membranes were incubated with secondary
antibody conjugated to horseradish peroxidase at 37°C for 30 min. Finally, the immunoreactive bands were visualized using a Super Signal West Pico Trial Kit (Pierce,
Rockford, Illinois, USA) according to the manufacturer’s instructions.
3
Western Blot Analysis of Myocardium Proteins
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Myocardium tissue was harvested for Western blot as described previously [23 (link)]. Cells of each group were harvested at appropriate time. Cells were washed three times with PBS and collected after ice-cold lysis buffer digestion. Protein lysates were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose (NC) membrane. Membranes were blocked with 5% milk in 1 × TBS-Tween-20 buffer and incubated overnight at 4°C with primary antibodies. Then, membranes were washed in Tris-buffered saline with Tween, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were developed using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) and visualized with UVP Bio-Imaging Systems. Blot densities were analyzed using ImageJ Software (National Institutes of Health, Bethesda, MD).
Primary antibodies are the following: anti-SIRT1, anti-IRS2, anti-P-Akt S473, anti-P-Akt T308, anti-t-Akt, anti-acetylated protein, anti-PGC-1α, anti-NRF1, anti-NRF2, anti-ERR-α, anti-TFAM, anti-GAPDH, and anti-β-actin (all from Abcam, Cambridge, MA, USA). Secondary antibodies are the following: horseradish peroxidase-conjugated goat anti-rabbit and goat anti-rat (from Zhongshan Biotechnology Co. Ltd., Beijing, China).
Primary antibodies are the following: anti-SIRT1, anti-IRS2, anti-P-Akt S473, anti-P-Akt T308, anti-t-Akt, anti-acetylated protein, anti-PGC-1α, anti-NRF1, anti-NRF2, anti-ERR-α, anti-TFAM, anti-GAPDH, and anti-β-actin (all from Abcam, Cambridge, MA, USA). Secondary antibodies are the following: horseradish peroxidase-conjugated goat anti-rabbit and goat anti-rat (from Zhongshan Biotechnology Co. Ltd., Beijing, China).
4
Protein Expression Analysis in Treated Cells
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Cells were treated with NT157 (0.3–1.3 μM) for 48 h or left untreated, and cell lysates were prepared and processed as previously described (30 (link)). The membranes were incubated overnight with the following primary antibodies: anti-Shc clone PG-797, anti GAPDH, anti-β-actin (Santa Cruz Biotechnology, San Diego, CA, USA), anti-phospho-Akt (Ser473) clone 736E11, anti-Akt, anti-ERK (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-ERK (Tyr202/Tyr204) (Covance, Princeton, NJ, USA), anti-IRS-1 (Upstate Biotechnology, Temecula, CA, USA), anti-IRS-2 (Abcam, Cambridge, UK), and phospho-IRS-1 (Tyr612) (Invitrogen, USA); anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA) were used as secondary antibodies.
5
Protein Expression Analysis in MIN6 Cells
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Total protein was extracted from MIN6 cells with cold RIPA lysis buffer containing proteases and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electrotransfer onto a PVDF membrane (Millipore, Billerica, MA, United States) and immunoblotting. Then, the membrane was blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h and incubated overnight at 4°C with anti-Glut4, anti-INS-R, anti-IRS-1, anti-IRS-2, and anti-GAPDH primary antibodies (1:1,000; Abcam, Cambridge, MA, United States). The membranes were washed three times in Tris-buffered saline and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; Gaithersburg, MD, United States) for 1 h. The expression of the specific protein was visualized using a commercial electrochemiluminescence kit (Invitrogen).
Ultra-performance liquid chromatography was coupled with time-of-flight mass spectrometry analysis (UPCL-TOF/MSE).
Ultra-performance liquid chromatography was coupled with time-of-flight mass spectrometry analysis (UPCL-TOF/MSE).
6
Insulin Signaling Pathway Protein Analysis
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Proteins were extracted from the skeletal muscle and adipose tissue by using lysis buffer. The proteins were then separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% or 15%) and electrophoretically transferred into polyvinylidene fluoride membranes. The membranes were probed with anti-InsR (1:20,000; Abcam, Cambridge, UK), anti-IRS-1 (1:500; Abcam, Cambridge, UK), anti-IRS-2 (1:5000; Abcam, Cambridge, UK), anti-GLUT-4 (1:2000; Abcam, Cambridge, UK) and anti-PPAR-γ (1:5000; Abcam, Cambridge, UK) antibodies overnight at 4 °C, and then incubated with a horse radish peroxidase (HRP)-coupled secondary antibody. Detection was performed using a ChemiDoc XRS+ (Bio-Rad, Hercules, CA) image analysis system.
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