WHS was prepared and standardized as described previously [25 (link)]. IBMX, DEX, insulin, oil red O, propidium iodide (PI), sodium orthovanadate, sodium fluoride (NaF), phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (PIC) were obtained from Sigma Aldrich Inc. (St Louis, MO, USA). Total RNA extraction kit (easy-BLUE™) and protein lysis buffer (PRO-PREP™) were purchased from iNtRON Biotechnology (Seongnam, Korea). Antibodies of ACC, p-AMPKα, AMPKα, p-Akt, p-Akt2, p-mTOR, mTOR, C/EBPα, p27, and SIRT-1 were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Akt, cyclin D1, cyclin E, cyclin B1, CDK2, FAS, FABP4, p21, PPARα, PPARγ, UCP-1, and β-actin antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). SREBP-1 antibody was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
P akt2
Manufactured by Cell Signaling Technology
Sourced in United States
P-Akt2 is a phosphorylated form of the Akt2 protein kinase. Akt2 is a serine/threonine-protein kinase that plays a key role in the insulin signaling pathway and is involved in the regulation of glucose metabolism, cell proliferation, and cell survival.
Automatically generated - may contain errors
Lab products found in correlation
P akt1, by Cell Signaling Technology (3 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
P gsk3β, by Cell Signaling Technology (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Sodium fluoride, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
P ampkα, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Pparα, by Santa Cruz Biotechnology (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
6 protocols using p akt2
1
Adipogenic Differentiation Pathway Analysis
2
Western Blot Analysis of Bone Signaling Proteins
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Subchondral bone tissues or RAW 264.7 cells were lysed in RIPA buffer (Solarbio) supplemented with PMSF, a phosphatase inhibitor, and protease inhibitor (Sigma-Aldrich). The lysates were centrifuged at 4°C, 12,000g for 20 minutes. Total protein concentration was measured by bicinchoninic acid Protein Assay Kit (Thermo Scientific). The aliquot of proteins was subjected to SDS-PAGE and transferred electrophoretically to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat milk, the membranes were incubated with primary Ab, including p-Akt1 (1:1000; Cell Signaling Technology, 9018), Akt1 (1:1000; Cell Signaling Technology, 2938), p-Akt2 (1:1000, Cell Signaling Technology, 8599), Akt2 (1:1000, Cell Signaling Technology, 3063), CTSK (1:1000; Abcam, ab19027), RANKL (1:1000; Abcam, ab62516), OPG (1:1000; ABclonal, A13250), and β-actin (1:10000; Proteintech, 66009-1-lg). After washing, the blots were probed with HRP-conjugated secondary Ab (1:10,000; Cell Signaling Technology) and subjected to enhanced chemiluminescence detection (Thermo Fisher Scientific). The intensity of bands was quantified by ImageJ and normalized to the density of the internal control.
3
Synthesis and Characterization of PBT-6
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Primary antibodies against PI3KC2γ (Thermo Fisher Scientific, Rockford, IL, USA), p-AKT1, p-AKT2, p-mTOR, β-actin (Cell Signaling Technology, Danvers, MA, USA), RANKL (Abcam, Cambridge, MA, UK), and NFATc1 (Santa Cruz Biotechnology, Dallas, CA, USA) were purchased. RANKL and M-CSF were obtained from R&D Systems (Minneapolis, MN, USA). In addition, method of PBT-6 synthesis was in detail described in supplementary method. In brief, the first step was the acetylation of NH2 under mild conditions with pyridine and acetic anhydride. In the second step, bromo benzothiazol and boronic acid were coupled via the Suzuki reaction using a phosphine ligand, base, and Pd catalyst. In the final step, it was hydrolyzed in NaOH solution. 6-(pyridin-4-yl) benzo[d] thiazol-2-amine: 1H NMR (400 MHz, DMSO): 8.58 (d, J=6, 2H-Ar), 8.18 (d, J=1.6, 1H-Ar), 7.70-7.66 (m, 5H), 7.42 (d, J=8, 1H-Ar).
4
Western Blot Protocol for AKT2, Cadherins
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The cells were lysed with buffer containing 2% sodium dodecyl sulfate, 50 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol, 10% glycerol, 0.002% bromophenol blue, and protease inhibitor mixture (Roche, Basel, Switzerland). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocking in 5% nonfat milk or bovine serum albumin, the membrane was immunoblotted with antibodies and visualized with horseradish peroxidase-coupled secondary antibodies. The primary antibodies against AKT2, p-AKT2, cyclin D1, E-cadherin, N-cadherin, and β-actin were from Cell Signaling Technology (Danvers, MA, USA).
5
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Total lysates from cells under the different experimental conditions were separated on 7.5% polyacrylamide denaturing gels and blotted onto nitrocellulose membranes (GE Healthcare Life Science, Little Chalfont, United Kingdom), which were reacted with antibodies directed against E-cadherin (sc-7870), Vimentin (sc-373717), and β-Catenin (sc-7963) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); against Akt2 (#3063), p-Akt2 (Ser474, #8599), p-GSK-3β (Ser9, #5558), GSK-3β (#9315), p-p70 S6 Kinase (Thr389, #9205), and p70 S6 Kinase (#9202) from Cell Signaling Technology (Danvers, MA, USA); and against Akt1 (#610860, BD Biosciences), anti-p-Akt1 (Ser473, #05-739), and anti-β-Tubulin (#T4026), following previously reported procedures [27 (link)]. The immunocomplexes were detected by using a WESTAR NOVA 2.0 (Cyanagen, Bologna, Italy), and the chemiluminescence-derived bands were captured with an ImageQuantTM LAS 4000 imager (GE Healthcare Life Science) and quantified with Image Quant TL software v7.0 (GE Healthcare Life Science), as previously reported [27 (link)].
6
Cardiac Tissue Protein Expression Analysis
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Cardiac tissue was homogenized with RIPA lysis buffer to prepare the lysate. Protein samples were subjected to SDS‐PAGE and then transferred to PVDF membranes (Bio‐Rad, Hercules, CA). After blocking with 5% BSA (Bovine serum albumin), the membrane was incubated with the antibody. The primary antibodies used include Fibronectin (FN), TGF‐β, ICAM‐1, VCAM‐1, IL‐1β, all of which were purchased from Abcam, Cambridge, MA. Phospho (p)‐t‐Akt, total (t‐)Akt, p‐Akt1, Akt1, p‐Akt2, Akt2, p‐GSK‐3β, GSK‐3β, p‐GS, GS, p‐AS160, AS160, p‐ERK1/2, ERK1/2, hexokinase II (HK II), all of which were purchased from Cell Signaling Technology, Beverly, MA. COL1A1, β‐Actin, all of which were purchased from Santa Cruz Biotech. Inc 3‐NT was purchased from Millipore. 4‐HNE was purchased from Alpha Diagnostic. Inc p‐PFKFB2, PFKFB2 and glycogen phosphorylase (GP) were purchased from Thermo Fisher Scientific, Waltham, MA. Antibody against MT was purchased from DakoCytomation, Santa Clara, CA. The ratios of total protein of Akt2, t‐Akt, Akt1, GSK‐3β, GS, AS160, PFKFB2 and ERK1/2 to β‐Actin are in the supplementary figures.
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