Signaling through Akt and mTOR was assessed in response to insulin in CON and IUGR myoblasts. Myoblasts were plated at 105 per well in 6 well plates and incubated in SkGM™ and 5% FBS for 24 h (or until cells were 70% confluent). Myoblasts were serum starved for 16 h in DMEM and exposed to insulin at 0, 1, and 10 nM concentrations for 10, 30, and 60 min. Myoblasts were washed with ice-cold PBS and lysis buffer was added (50μL per well; 20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2.5 mM Na pyrophosphate, 20 mM NaF) with 0.004% v/v phosphatase inhibitor II/III (Sigma-Aldrich) and 0.01% v/v protease inhibitor (Sigma-Aldrich). Lysate was centrifuged at 15,000 rpm for 10 min and supernatant was stored at −70 C. Protein quantification, gel electrophoresis, and transfer to nitrocellulose membranes was performed as previously reported (Brown et al. 2012 (link)). Membranes were incubated with the following primary rabbit polyclonal IgG antibodies: phospho-AKT(S473), total AKT, phospho-p44/42 (Thr202/Tyr204) extracellular signal-regulated kinase (ERK1/2), total p44/42 ERK1/2, phospho-p70S6K (Thr389), total p70S6K, phospho-mTOR (S2448), and total mTOR (1:1000; Cell Signaling Technology). Actin mouse monoclonal IgG (1:100,000; MP Biomedicals, Santa Ana, CA) was used as a loading control.
Phosphatase inhibitor 2 3
Manufactured by Merck Group
Phosphatase inhibitor II/III is a laboratory reagent used to inhibit the activity of phosphatases, a class of enzymes that remove phosphate groups from proteins. This inhibitor is commonly used in protein analysis and purification procedures to prevent the dephosphorylation of proteins, which can interfere with the study of protein function and signaling pathways.
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2 protocols using phosphatase inhibitor 2 3
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Signaling Pathways in IUGR Myoblasts
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Detailed Cellular Lysis and Actin Buffers
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Lysis buffer: 25 mM HEPES, pH7.4, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail (Roche), phosphatase inhibitor II, III (Sigma).
HTG buffer: 50 mM HEPES, pH7.5, 150 mM NaCl, 1% Trition X-100, 10% glycerol.
Harsh HTG buffer: 20 mM Tris pH7, 500 mM NaCl, 5 mM imidazole, 2 mM MgCl2, 1% Triton, 1 mM DTT. G-actin buffer: 5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2.
Actin polymerization buffer: 10 mM Tris, pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM ATP.
HBS: 25 mM HEPES, pH7.5, 137 mM NaCl, 5 mM KCl, 15 mM glucose, 1.5 mM CaCl2, 0.8 mM MgCl2.
HTG buffer: 50 mM HEPES, pH7.5, 150 mM NaCl, 1% Trition X-100, 10% glycerol.
Harsh HTG buffer: 20 mM Tris pH7, 500 mM NaCl, 5 mM imidazole, 2 mM MgCl2, 1% Triton, 1 mM DTT. G-actin buffer: 5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2.
Actin polymerization buffer: 10 mM Tris, pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM ATP.
HBS: 25 mM HEPES, pH7.5, 137 mM NaCl, 5 mM KCl, 15 mM glucose, 1.5 mM CaCl2, 0.8 mM MgCl2.
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