Mice were injected subcutaneously with either vehicle, native human insulin (Novo Nordisk, Malov, Denmark) or recombinant human IGF-1 (Ipsen, Slough, UK). Dosage was calculated based on the average weight of all lean mice (assuming blood volume does not significantly alter in obese mice). Plasma levels of human IGF-1 and human insulin in the mice were measured using ELISAs (insulin; Novo Nordisk, Malov, Denmark. IGF-1; Immunodiagnostic Systems, Tyne & Wear, UK) as described previously,14 (link) in order to confirm equivalent dosing levels between HF and LF mice. After 15 min stimulation, mice were euthanised and the aorta rapidly harvested and snap-frozen. Twenty micrograms of protein was processed for Western blotting. Nitrocellulose membranes were probed with antibodies diluted in 5% BSA; 1:1000 Akt, 1:2000 phosphorylated Akt (Ser473) and 1:20,000 beta actin (Cell Signaling).
Phosphorylated akt ser473
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphorylated Akt (Ser473) is a laboratory reagent that detects the phosphorylation of Akt at serine 473. Akt is a key signaling protein involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (9 mentions)
β actin, by Merck Group (8 mentions)
β actin, by Santa Cruz Biotechnology (7 mentions)
Total akt, by Cell Signaling Technology (6 mentions)
Hybond, by Cytiva (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Las 1000, by Fujifilm (3 mentions)
Ecl plus reagent, by GE Healthcare (3 mentions)
Hybond p, by GE Healthcare (3 mentions)
Protease inhibitor cocktail, by Takara Bio (3 mentions)
Ripa buffer, by Fujifilm (3 mentions)
Phosphorylated erk thr202 tyr204, by Cell Signaling Technology (3 mentions)
Caspase 7, by Cell Signaling Technology (3 mentions)
Erk1 2, by Cell Signaling Technology (3 mentions)
Phosphorylated erk1 2, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Phosphorylated enosser1177, by Cell Signaling Technology (2 mentions)
64 protocols using phosphorylated akt ser473
1
Insulin and IGF-1 Stimulation in Mice
2
Western Blot Analysis of SULF1 and Akt
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The tumor cells were lysed with cell lysis buffer containing 25 μg protein, separated by 4–12% gradient NuPAGE gel (Invitrogen, Carlsbad, CA) and transferred onto polyvinylidene difluoride membranes (Amersham, Bioscience, Buckinghamshire, UK). The membranes were probed with antibodies at 4°C overnight against SULF1 (1:1000, H-41, Santa Cruz), Akt (1:1000, Cell Signaling Technology), phosphorylated AktSer473 (1:1000, Cell Signaling Technology) and GAPDH as a loading control (6C5, 1:10,000, Millipore) after blocking with 5% skimmed milk in TBST buffer at room temperature for 1 hour. The secondary antibody was incubated at room temperature for 1.5 hours, and proteins were visualized by a chemiluminescence system (Amersham Biosciences).
3
Western Blot Analysis of CCK-BR-Mediated Signaling
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Cellular proteins were extracted 24 h after treatment with CCKBR APs (100 nM). Protein concentration was determined using a MicroBCA assay (Pierce), and cell lysates (60 μg of protein) were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, blocked in 5% BSA, and incubated overnight (4°C) with primary antibodies. Antibodies used were as follows: phosphorylated-Akt (Ser473) (#4060; Cell Signaling Technology), total Akt (#4691; Cell Signaling Technology), and beta-actin (#A2228; Sigma). The blots were washed and probed with secondary antibody coupled to horseradish peroxidase (HRP; Amersham), and HRP activity was detected using an enhanced chemiluminescent substrate (Pierce).
4
Quantitative Western Blot Analysis
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Western blot analysis was conducted according to a previous study [25] (link). Briefly, the equal amounts of proteins obtained from the ileum and jejunum samples were separated by a reducing SDS-PAGE electrophoresis. The proteins were transferred onto PVDF membranes (Millipore, MA, USA) and blocked with 5% non-fat milk in Tris-Tween buffered saline buffer (20 mM Tris, pH 7.5,150 mM NaCl, and 0.1% Tween-20) for 3 hr. The following primary antibodies were incubated overnight at 4°C with gentle rocking: β-actin (Santa Vruz, 1∶400), total 4EBP1 (T-4EBP1, Cell Signaling, 1∶1000), phosphorylated 4EBP1 (Ser 65) (Cell Signaling, 1∶1000), total Akt (T-Akt, Cell Signaling, 1∶1000), phosphorylated Akt (Ser 473) (Cell Signaling, 1∶1000), total mTOR (T-mTOR, Cell Signaling, 1∶1000) or phosphorylated mTOR (Ser 2448) (Cell Signaling, 1∶1000). Then, the HRP-conjugated secondary antibodies were subsequently incubated for 1 hr at room temperature before developing the blots using Alpha Imager 2200 software (Alpha Innotech Corporation, CA, USA). We digitally quantified the resultant signals and normalized the data to the actin abundance. β-actin was used as an internal loading control for cytoplasmic protein fractions.
5
Western Blot Analysis of eNOS Phosphorylation
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Cell lysates were prepared using RIPA buffer (Wako Pure Chemical Industries, Ltd.) containing a protease inhibitor cocktail (Takara Bio Inc.) and phosphatase inhibitors (Roche). Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Hybond-P; GE Healthcare). After blocking with 5% bovine serum albumin, the membranes were incubated with primary antibody against either phosphorylated-eNOSSer1177, Akt, phosphorylated-AktSer473 (Cell Signaling Technology), eNOS (BD Biosciences), or β-actin (Sigma) overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse Ig (Cell Signaling Technology) or anti-rabbit Ig (Chemicon) antibody was then used as the secondary antibody. Antibody distribution was visualized with ECL-plus reagent (GE Healthcare) using a luminescent image analyzer (LAS-1000, Fuji Film).
6
Hippocampal Protein Signaling Assay
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The hippocampus samples were lysed in 20 mM Tris buffer (pH 7.4) containing 2 mM EDTA, 137 mM NaCl, 1% NP40, 10% glycerol, and 12 mM α-glycerol phosphate and protease inhibitors. After 30 min on ice, the lysates were centrifuged for 10 min at 11,300× g at 4 °C. After measuring the protein contents in the lysates using a Bio-Rad protein assay kit, lysates with equal amounts of protein were immunoprecipitated with specific antibodies before separation by SDS-PAGE as previously described [13 (link)]. The antibodies used for immunoblot analysis were cAMP responding element-binding protein (CREB), phosphorylated CREBser133, Akt, phosphorylated AktSer473, GSK-3β, phosphorylated GSK-3βser9, tau, phosphorylated tauser396, and β-actin (Cell Signaling Technology). The intensity of protein expression was determined using Imagequant TL (Amersham Biosciences, Little Chalfont, England).
7
Aortic and endothelial protein analysis
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Aortic tissue and HAECs homogenates (10 to 25 μg) were separated via SDS-PAGE and transferred to Immobilon-P poly(vinylidene fluoride) membranes. Immunoblots were probed with antibodies for PTP1B (mouse: Abcam; human: BD Bioscience), GAPDH (Santa Cruz), COX-1 (Abcam), COX-2 (BD Bioscience), endothelial NO synthase (BD Bioscience), and phosphorylated endothelial NO synthase 1177/79 (BD Bioscience), Akt, and phosphorylated Akt (Ser473, Cell Signaling).
8
Western Blot Analysis of Muscle Proteins
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The extraction of cellular protein and western blot analysis were performed as previously described.22 Cell lysates were separated on 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrotransferred onto polyvinylidene difluoride membrane. The membranes were blocked with skim milk (5%) for 1 h and then incubated with the primary antibodies for Akt, phosphorylated Akt (Ser473) (Cell Signaling, Danvers, MA, USA), myogenin, MHC, β‐actin (Santa Cruz, Santa Cruz, CA, USA), and acrolein protein adduct (Novus Biologicals, Littleton, CO, USA) overnight at 4°C. The membranes were incubated with anti‐rabbit or anti‐mouse antibodies conjugated to horseradish peroxidase for 1 h. The blots were visualized with enhanced chemiluminescence reagent (BioRad, Hercules, CA, USA) and exposed to X‐ray film. The densitometric analysis was assessed using ImageJ software.
9
Western Blot Analysis of Signaling Pathways
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Cells were seeded in 60-mm dishes and treated with AZD6738, cytotoxic agents, or AZD6738 plus cytotoxic agents for 5 days. The cells were harvested and lysed in RIPA buffer containing protease inhibitors on ice for 30 minutes. The proteins were extracted and equal amounts of proteins were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by western blot analyses. Primary anti-bodies against the following molecules were purchased from Cell Signaling Technology (Beverley, MA): p53 (#9282); ATR (#2790); phosphorylated ATR-Ser428 (#2853); Chk1 (#2360); phosphorylated Chk1-Ser345 (#2341); phosphorylated AKT-Ser473 (#9271); AKT (#9272); phosphorylated ERK-Thr202/Tyr204 (#9101); ERK (#9102); PARP (#9532); caspase-7 (#9492); phosphorylated CDC2 (#9111); CDC2 (#9112); and p21 (#2947). α-Tubulin (#T5168) and β-actin antibodies were purchased from Sigma-Aldrich. Anti-ATM antibody (#ab78) was obtained from Abcam Bioscience (Cambridge, UK). Anti-γH2AX antibody (#05-636) was bought from Millipore (Billerica, MA). Anti-permeability glycoprotein (p-glycoprotein) and the secondary antibodies were purchased from Thermo Scientific (Waltham, MA). The intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
10
Protein Expression Analysis in Muscle Tissues
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Muscle tissues were homogenized in RIPA buffer containing a protease inhibitor cocktail (Santa Cruz, Dallas, TX) and phosphatase inhibitor (Research Product International, Mount Prospect, IL). Protein concentration of samples were determined by a standard BCA assay and the samples were subjected to standard western blot protocol, as described previously (Hartnett et al. 2015 ). Proteins that were transferred onto the membranes were immunoblotted using the following primary antibodies: AKT, phosphorylated AKT (Ser473), FoxO3, phosphorylated FoxO3 (Ser253), LC3, IRS1, AMPKα and phosphorylated AMPKα (Thr172) (Cell Signaling Technologies, Danvers, MA); GAPDH, Ubiquitin, pERK and ERK (Santa Cruz Inc., Santa Cruz, CA); MurF1 and Atrogin1 (GeneTex, Irvine, CA); and PGC1α (Millipore‐Sigma, St. Louis, MO). The secondary antibodies conjugated with Alex‐700 or ‐800 fluorescence were purchased from Invitrogen (Thermo Fisher Scientific Inc., Carlsbad, CA). The fluorescent signals of the blots were detected by LI‐COR scanner (LI‐COR Biosciences, Lincoln, NE) and quantified using LI‐COR Image Studio.
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