All rats were sacrificed at 12 wk postoperatively. Livers were sampled and immediately frozen in liquid nitrogen and stored at -80 °C until analysis. Alterations in the insulin signaling pathway, as indicated by the protein expression of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), insulin receptor substrate 2 (IRS-2), PI3K, and AKT were determined by Western blotting. Samples were mechanically dissociated and lysed in radioimmunoprecipitation assay 37 (RIPA) buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L Na2-EDTA, 1% NP-40, 0.25% Na-deoxycholate) containing protease and phosphatase inhibitor cocktails (Roche, United States). Following brief sonication and heating, the supernatants were subjected to SDS-PAGE and transferred to PVDF membranes. Blots were incubated overnight at 4 °C with primary antibodies (anti-insulin receptor antibody; anti-IRS1 antibody; anti-IRS2 antibody; anti-PI3K P85 alpha antibody; anti-pan-AKT antibody; anti-beta-actin antibody, all from Abcam) and were then incubated with secondary antibodies (Abcam). Blots were visualized with an enhanced chemiluminescence reagent (Millipore) and quantified with Image Lab (Bio-Rad).
Anti pan akt antibody
Manufactured by Abcam
Sourced in United Kingdom, China, United States
The Anti-pan-AKT antibody is a primary antibody that recognizes all three isoforms of the AKT (Protein Kinase B) serine/threonine protein kinase. AKT is a key regulator of cellular processes such as cell proliferation, growth, survival, and metabolism. This antibody can be used to detect and analyze the expression levels of AKT in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Image lab, by Bio-Rad (3 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Anti insulin receptor antibody, by Abcam (2 mentions)
Anti irs1 antibody, by Abcam (2 mentions)
Anti irs2 antibody, by Abcam (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Ctla 4 antibody, by Abcam (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Diethylnitrosamine, by Merck Group (1 mentions)
β actin antibody, by Abcam (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Ab8805, by Abcam (1 mentions)
A6058, by ABclonal (1 mentions)
1 step nbt bcip substrate solution, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab81283, by Abcam (1 mentions)
Monoclonal anti β actin peroxidase antibody, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
6 protocols using anti pan akt antibody
1
Insulin Signaling Pathway Alterations
2
Diethylnitrosamine-Induced Liver Injury Model
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Diethylnitrosamine (DEN) was purchased from Sigma (N0258, Sigma-Aldrich, USA). Carbon tetrachloride was purchased from Hao-Sen Chemical Co., Ltd. (Shanghai, China). Ethanol (55%) was purchased from Niu Lan Shan Co., Ltd. (Beijing, China). The TRI Reagent was purchased from Sigma (T9424; Sigma-Aldrich, USA). The Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher (K1621, Thermo Fisher; U.S.A). HieffTM qPCR SYBR® Green Master Mix was purchased from Yeasen Biotech Co., Ltd. (Cat. No. 11203, Shanghai, China). Anti-pan-AKT antibody (#ab8805, Abcam, Cambridge, U.K.), anti-p-AKT (ser473) antibody (#ab126433, Abcam, Cambridge, U.K.), Anti-AFP primary antibody (cat# ab46799, Abcam, Cambridge, U.K.), β-actin antibody (cat# ab8227, Abcam, Cambridge, U.K.), anti-IL-10 primary antibody (cat#ab189392, Abcam, Cambridge, U.K.), and CTLA-4 antibody (cat#ab134090, Abcam, Cambridge, U.K.) were purchased from Abcam (UK). CD4 (cat#25229), CD8α (cat#98941), and PD-1 (cat#84651) antibodies were purchased from Cell Signaling Technology (M.A. USA).
3
Quantifying SEMA6C Protein Expression
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To detect the effect of Si‐Sema6c on SEMA6C protein expression, the ovaries were harvested after 48 hrs culture. While the rest of ovaries of each group were lysed in RIPA buffer after 4 days culture for next Western blotting experiments as the routine procedure, membranes were incubated with different primary antibody overnight at 4 °C: anti‐SEMA6C (AF2108, 1:800, 0.25 μg/ml; R&D Systems), AKT (anti‐pan‐AKT antibody, Abcam, ab8805, 1:1000), p‐AKT (phospho‐S473, Abcam, ab81283, 1:1000), rpS6 (ribosomal protein S6) polyclonal antibody (A6058, 1:1000; ABclonal, Wuhan, China), phospho‐rpS6 (S235/236, AP0296, 1:1000; ABclonal) and visualized by Pierce™ ECL Western blotting substrate (No. 32209; Thermo Fisher Scientific, Waltham, MA, USA) or AP (alkaline phosphatase) substrate (1‐Step™ NBT/BCIP substrate solution, No. 34042: Thermo Fisher). Integrated light intensity of each band was quantified with Image Lab (Java image processing software, BioRad, Hercules, CA, USA) and used to compare treatment‐induced changes in the concentration of phosphoproteins. GAPDH expression was measured to verify equal loading.
4
Quantitative Western Blot Analysis
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Western blot analysis was performed as previously described [8 (link),9 (link)]. Briefly, arteries were homogenized, and 30 μg protein was loaded in each lane. A mouse monoclonal antibody against neuronal nitric oxide synthase (nNOS, 1:2000, BD Biosciences), a rabbit polyclonal anti-pan-AKT antibody (1:1000, Abcam), or a rabbit polyclonal anti-pan-AKT (phospho T308, 1:500, Abcam) were used. The development and quantification of the images were performed using Quantity One software (Windows v4.6.6, Bio-Rad, Madrid, Spain). The same membrane was used to correct protein expression in each sample, by means of a monoclonal anti-β-actin−peroxidase antibody (1:50,000; Sigma-Aldrich, Madrid, Spain).
5
Protein Expression in Insulin Signaling
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All rats were sacrificed at 12 wk postoperatively. Liver and skeletal muscle were sampled and immediately frozen in liquid nitrogen and stored at -80 °C until analysis. Alterations of the insulin signaling pathway were determined by Western blotting, as indicated by protein expression of insulin receptor, insulin receptor substrate (IRS)1, IRS2, phosphatidylinositol 3-kinase (PI3K) and Akt. For Western blotting, samples were mechanically dissociated and lysed in radioimmunoprecipitation assay 37 (RIPA) buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L Na2-EDTA, 1% NP-40, 0.25% Na-deoxycholate) containing protease and phosphatase inhibitor cocktails (Roche, United States). After brief sonication and heating, the supernatants were subjected to SDS-PAGE and transferred to PVDF membranes. Blots were incubated overnight at 4 °C with primary antibodies (anti-insulin receptor antibody, anti-IRS1 antibody, anti-IRS2 antibody, anti-PI3K P85 α antibody, anti-pan-AKT antibody, anti-β-actin antibody; all Abcam Cambridge, MA, United States) and were then incubated with secondary antibodies (Abcam). Blots were visualized with an enhanced chemiluminescence reagent (Millipore, Billerica, MA, United States) and quantified with Image Lab (Bio-Rad, Hercules, CA, United States).
6
Western Blot Analysis of Signaling Proteins
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Western blot analysis was performed as previously described34 (link). For these experiments, 20 μg protein were loaded in each lane. We used a monoclonal purified mouse anti-eNOS/NOS Type III antibody (1:2500; BD Biosciences), a rabbit polyclonal antibody against eNOS phosphorylated in Ser1177 (P-eNOS, 1:2000; Abcam), a mouse monoclonal antibody against iNOS (1:5000 dilution; Transduction Laboratories), a mouse monoclonal anti-soluble guanylate cyclase –alpha1(sGC-α1) subunit antibody (1:500; Santa Cruz), a mouse monoclonal anti-soluble guanylate cyclase –beta1 (sGC-β1) subunit antibody (1:500; Santa Cruz), a mouse monoclonal anti-alpha 1 adrenergic receptor antibody (1:1000; Abcam), a rabbit polyclonal anti-PI 3 Kinase p85 beta antibody (1:2000; Abcam), a rabbit polyclonal anti-pan-AKT antibody (1:500; Abcam), a rabbit polyclonal anti-pan-AKT (phospho T308) antibody (P-AKT, 1:500; Abcam), and a monoclonal anti-β-actin-peroxidase antibody (1:50000; Sigma-Aldrich). Appropriate positive controls (+C) were used for each analysis (see figure legends).
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