Total proteins were extracted from homogenized tissues and cell lysates using RIPA buffer containing protease inhibitors and phosphatase inhibitors (Pierce, Rockford, USA). An equal amount of protein lysates was separated on 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After an overnight incubation with the following primary antibodies at 4 °C: HOXB5 (1:1000, #109375, Abcam, Cambridge, UK), SESN3 (1:800, #97792, Abcam), IGF1R (0.1 μg/mL, #AF-305-SP, R&D Systems, Inc., Minneapolis, USA), protein kinase B (Akt, 1:1000, #9272, Cell Signaling), phosphorylated Akt (p-Akt, 1:1000, #9271, Cell Signaling, Danvers, USA), PCNA (PCNA, 1:1000, #18197, Abcam), p27 (1:1000, #32034, Abcam), B-cell lymphoma 2 (Bcl-2, 1:800, #59348, Abcam), E-cadherin (1:2000, #40772, Abcam), N-cadherin (1:1000, #76057, Abcam), cleaved caspase-3 (1:1000, #49822, Abcam), and GAPDH (1:2000, #37168, Abcam), the membranes were washed with Tris-Buffered Saline and stained with secondary antibody (1:2000, #6721, Abcam) for 60 min at room temperature. The images were captured using Alphalmager™ 2000 Imaging System (Alpha Innotech, USA).
Phosphorylated akt p akt
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Phosphorylated Akt (p-Akt) is a lab equipment product that detects the phosphorylated form of the protein Akt, also known as protein kinase B. Akt is a key regulator of cellular processes such as cell growth, proliferation, and survival. The phosphorylation of Akt is a critical step in the activation of this signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Phosphorylated erk1 2 p erk1 2, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Total akt t akt, by Cell Signaling Technology (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Anti cd9, by System Biosciences (3 mentions)
Anti cd63, by System Biosciences (3 mentions)
Anti cd81, by System Biosciences (3 mentions)
Gapdh, by Abcam (2 mentions)
P pi3k, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Histone h3, by Abcam (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Abcam (2 mentions)
33 protocols using phosphorylated akt p akt
1
Western Blot Analysis of Cellular Proteins
2
Reagents and Antibodies Used in Cellular Assays
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The reagents used in the present study were purchased from the following suppliers: FR and API-1 from Tocris Bioscience (Bristol, UK); RPMI-1640 medium, fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA); water soluble tetrazolium-1 (WST-1), Cytotoxicity Detection Kit Plus, Cell Proliferation ELISA colorimetric kit and Cell Death Detection ELISA Plus kit from Roche Diagnostics GmbH (Mannheim, Germany); and PathScan ® Cleaved Caspase-3 (Asp175) Sandwich ELISA kit and monoclonal rabbit antibodies against β-actin (ACTB; catalog no., 4970; dilution, 1:1,000), B-cell lymphoma-2 (BCL2)-associated X protein (BAX; catalog no., 5023; dilution, 1:1,000), BCL2 antagonist/killer (BAK; catalog no., 12105; dilution, 1:1,000), cyclin D1 (CYCD1; catalog no., 2978; dilution, 1:1,000), cMYC (catalog no., 13987; dilution, 1:1,000), Akt (catalog no., 4685; dilution, 1:1,000), ERK1/2 (catalog no., 4370; 1:2,000), phosphorylated Akt (pAkt; catalog no., 4060; dilution, 1:2,000), phosphorylated ERK1/2 (pERK1/2; catalog no., 4094; dilution, 1:1,000) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (catalog no., 7074; dilution, 1,1000) were provided by Cell Signaling Technology (Danvers, MA, USA). All other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
3
Adipocyte Protein Analysis via Western Blot
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The adipocytes, after treatments with AS-IV, CTRP3 siRNA or wortmannin, were used for protein extraction with RIPA buffer containing the protease inhibitor cocktail (Sigma). The protein concentrations of the extracted samples were determined by bicinchoninic acid protein assay kit (Thermal Fisher Scientific). Equal amounts of proteins (30 µg) were resolved on 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Then, PVDF membranes were blocked with 6% skimmed milk for 1.5 h followed with incubation by antibodies against phosphorylated PI3K (p-PI3K; 1:1000, Cell Signaling Technology), PI3K (1:1000, Cell Signaling Technology), phosphorylated AKT (p-AKT; 1:1000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology), CTRP3 and β-actin (1:3000, Cell Signaling Technology). Membranes were probed with corresponding horseradish peroxidase-conjugated secondary antibodies (1:2000; Cell Signaling Technology). The bands were detected by the Enhanced Chemiluminescence Kit (Thermo Fisher Scientific). Protein levels were analyzed using ImageJ software using β-actin for normalization.
4
Evaluating Oxidative Stress and Apoptosis
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Cell counting kit-8 (CCK-8) and Annexin V-FITC/PI apoptosis detection kits were purchased from Vazyme Biotechnology Company (Nanjing, China). T-BHP, dimethyl sulfoxide (DMSO), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin and N-acetylcysteine (NAC) were purchased from Macklin Biochemical Corporation (Shanghai, China). CpG ODN1826 was purchased from InvivoGen (San Diego, CA, USA). The JC-1 kit was purchased from Beyotime (Shanghai, China). RAW264.7 and AML12 (alpha mouse liver 12) cells were received from American Type Culture Collection (Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and Opti-MEM were purchased from Gibco (Waltham, MA, USA). Antibodies for cleaved-caspase 3, caspase 3, caspase 8, cleaved-caspase 8, caspase 9, cleaved-caspase 9, PARP, cleaved PARP, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated Akt (p-Akt), phosphorylated p38 MAPK, p38 MAPK, phosphorylated JNK, JNK, anti-rabbit IgG, anti-mouse IgG, and GAPDH were all purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NOX2 antibody (ab80508) was purchased from Abcam (Cambridge, MA, USA). AST and ALT detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
5
Western Blot Protein Analysis
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To isolate the proteins, cells collected from six-well plates were washed once in phosphate-buffered saline and lysed in RIPA buffer. Each protein sample (12 μg) was resolved by SDS–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and incubated with monoclonal antibodies, as follows: TAZ/YAP (1:1000; Cell Signaling, Danvers, MA, USA), N-cadherin (1:1000; clone 56, BD Biosciences, Tokyo, Japan), E-cadherin (1:1000; Cell Signaling), phosphorylated-AKT (p-AKT; 1:1000; Cell Signaling), AKT (1:1000; Cell Signaling), phosphorylated-ERK1/2 (p-ERK1/2; 1:1000; Cell Signaling), ERK1/2 (1:1000; Cell Signaling), phosphorylated β-catenin (p-β-catenin; 1:1000; Cell Signaling), β-catenin (1:1000; Cell Signaling), β-actin (1:2000; Cell Signaling), α-tubulin (1:20 000; Abcam, Cambridge, MA, USA), or histone H3 (1:20 000; Abcam). The signals were detected by incubation with secondary antibodies labelled with the ECL Detection System (GE Healthcare, Little Chalfont, UK).
6
Investigating Signaling Pathways in Cells
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The chemical LY294002 with DMSO as solvent was purchased from Selleck, TX, USA. The primary antibodies were purchased from Cell Signaling Technology (MA, USA), including AKT, phosphorylated AKT (p-AKT), caspase 3, caspase 9 and β-actin. The NUPR1 antibody was purchased from Abcam (Cambridge, UK). The primary antibodies of Bcl-2, Bax, Bcl-xl, CDK4 and CDK6 were purchased from Santa Cruz (CA, USA).
7
Lipid Metabolism Regulation Protocol
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RSL3 (#SML2234), aspirin (#A2093), OA (#O1383), POA (#P9417), PA (#P0500) and SA (#S4751) were purchased from Sigma-Aldrich (Darmstadt, Germany). Ferrostatin-1 (Fer-1, #HY100579), Z-VAD-FMK (#HY16658B), 3-Methyladenine (3-MA, #HY19312), MHY1485 (#HY-B0795) and Rapamycin (#HY10219) were obtained from MedChemExpress (NJ, USA). Primary antibodies against AKT (#4691), phosphorylated AKT (p-Akt; #4060), mTOR (#2983), phosphorylated mTOR (p-mTOR; #5536), p70S6K (#2708), phosphorylated p70S6K (p-p70S6K; #9204), 4E-BP1 (#9644), phosphorylated 4E-BP1 (p-4E-BP1; #2855) and SCD1 (#2794) were purchased from Cell Signaling Technology (MA, USA). SREBF1 antibody (#14088-1-AP) was obtained from Proteintech (IL, USA). The antibodies against Ki67 (#ab16667) and 4-HNE (#ab46545) were purchased from Abcam (MA, USA).
8
Western Blot Analysis of Notch Signaling
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A cell lysis buffer system (Santa Cruz) was used to prepare the whole-cell extracts on dry ice. The Nuclear Extraction Reagents (Pierce) and Total Protein Extraction kit (Beyotime) were used according to the protocol provided by the manufacturers. The protein concentrations were detected with a BCA protein assay kit (Pierce). Vertical SDS-PAGE was used to separate the protein samples, which were then transferred to the PVDF membranes. After blocking with 10% defatted milk, primary antibodies against Delta-like 1 (DLL1, Sigma-Aldrich, 1: 2000), DLL3 (Sigma-Aldrich, 1: 2000), DLL4 (Sigma-Aldrich, 1: 2000), Jagged1 (Abcam, 1: 2500), Jagged2 (Abcam, 1: 2500), NICD (Cell Signaling Tech, 1: 4000), PI3K (Cell Signaling Tech, 1: 2000), Akt (Cell Signaling Tech, 1: 2000), phosphorylated Akt (p-Akt, Cell Signaling Tech, 1: 2000), Bcl2 (Abcam, 1: 2500), active caspase3 (Abcam, 1: 2000), GAPDH (Abcam, 1: 2000), and Histone H3 (Abcam, 1: 2000) were incubated at 4°C for 8 h. Then, the secondary antibodies were used to incubate the membranes at room temperature for 1 h. An ECL kit (Pierce) was used to develop the membranes, which were then exposed with Gene Genius (Syngene). Image J software was used to analyze the densities of the blots.
9
Western Blot Analysis of Signaling Proteins
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Protein was extracted from cultured cells with cold lysis buffer containing phosphatase and protease inhibitor cocktail (Sigma). Total proteins were separated by electrophoresis with the 12% SDS-PAGE and then transferred onto nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA). The target proteins were measured using the primary antibodies of COX-2 (Abcam, Cambridge, United Kingdom), phosphorylated AKT (p-AKT, Cell Signaling, Danvers, MA), total AKT (t-AKT, Cell Signaling, Danvers, MA), NFκB-p65 (Abcam, Cambridge, United Kingdom), phosphorylated IκBα (p-IκBα, New England Biolabs, Beverly, MA), total IκBα (t-IκBα, New England Biolabs, Beverly, MA), and followed by detection with the secondary antibody and ECL kit (PerkinElmer, Waltham, MA). β-Actin (Santa Cruz, Dallas, TX) antibodies were used here as an internal control. Quantitative results are expressed as a ratio of target proteins to their internal control and phosphorylated proteins to their total proteins, accordingly.
10
Western Blot Analysis of Protein Phosphorylation
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Phosphatase inhibitor/buffer mixture II (Sigma-Aldrich, Munich, Germany) and protease inhibitor cocktail (Roche) were used to harvest the cells. They were resolved by sodium dodecyl sulfate–10 % polyacrylamide gel electrophoresis, and transferred to Immobilon-P membranes (Millipore, Schwalbach am Taunus, Germany). The membranes were then incubated with antibodies to activate phosphorylated ERK1 and ERK2 (p-ERK; Cell Signaling Technology, Danvers, MA, USA), total ERK1 and ERK2 (Cell Signaling Technology, Danvers, MA, USA), phosphorylated AKT (p-AKT; Cell Signaling Technology, Danvers, USA), and total AKT (Cell Signaling Technology, Danvers, USA). Antibodies were detected with the appropriate anti-mouse horseradish peroxidase conjugated secondary antibody enhanced by chemiluminescence (Pierce, Life Technologies, Darmstadt, Germany), and images were captured with a Versa Doc 5000 imaging system (Bio-Rad, Munich, Germany).
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