Intrathecal injection was performed as described previously.19 (link) Briefly, under anesthesia, laminectomy was performed at the L5 vertebra. PE-5 catheter was inserted into the subarachnoid space of the spinal cord at L4 level. The AKT inhibitor IV (0.8 µg/10 µL; Sigma-Aldrich) was administered on postoperative days 1, 3, and 5 in SNL + AKT inhibitor group. The SNL + vehicle group received 10 µL of 3% dimethyl sulfoxide in saline.
Akt inhibitor 4
Manufactured by Merck Group
Sourced in United States
AKT inhibitor IV is a laboratory reagent used in research applications. It functions as a selective inhibitor of the AKT serine/threonine protein kinase. AKT is an important signaling molecule involved in cellular processes such as metabolism, proliferation, cell survival, and angiogenesis. The AKT inhibitor IV can be used to study the role of AKT in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Trizol, by Qiagen (1 mentions)
Its liquid media supplement, by Merck Group (1 mentions)
Lv nc, by Genechem (1 mentions)
Pgcsil gfp vector, by Genechem (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
L tryptophan, by Merck Group (1 mentions)
Teriflunomide, by Bio-Techne (1 mentions)
L kynurenine, by Merck Group (1 mentions)
Human ifn γ, by Merck Group (1 mentions)
Amlexanox, by Bio-Techne (1 mentions)
N acetyl 5 methoxytryptamine, by Merck Group (1 mentions)
Aicar, by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Ns 398, by Merck Group (1 mentions)
Phenformin, by Merck Group (1 mentions)
Triacsin c, by Santa Cruz Biotechnology (1 mentions)
Antibiotic solution, by HiMedia (1 mentions)
15 protocols using akt inhibitor 4
1
AKT Inhibitor Intrathecal Injection in Neuropathic Pain
2
Cartilage Explant Culture and RNA Extraction
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For explant culture, cartilage tissues were obtained from 5 OA knees in pairs from preserved areas and degenerated areas on the tibial plateaus, as described for the cartilage protein analysis. After being rinsed in sterilized PBS, each piece of cartilage tissue was diced into 30–50 cubes of 2–3 mm. The diced cartilage, or explants, were equally divided into two, which were placed onto respective wells of a 12-well plastic plate. These explants were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with ITS Liquid Media Supplement (Sigma Aldrich, St. Louis, MO, USA) and 25 µg/ml ascorbic acid overnight, and then the media was replaced with those containing Akt inhibitor IV (Sigma Aldrich; 5 µΜ in dimethyl sulfoxide [DMSO]) or DMSO alone (vehicle control). After 48 h of culture, the explants were recovered, embedded in OCT compound, snap frozen in liquid nitrogen and stored at -80 °C until analysis. Extraction of RNA from the explants was performed following a previously described method [3 (link)]. In brief, 20 µµ-thick cryosections were prepared from the explants embedded in OCT compound, which were immediately immersed in TRIzol (Thermo Fisher Scientific). RNA was first recovered from the TRIzol reagent in the aqueous phase, and then purified using the RNeasy Micro kit (Qiagen).
3
Intra-Trigeminal Ganglion Microinjection
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Recombinant lentiviruses containing Cxcr3 shRNA (LV-Cxcr3 shRNA, 5′-CTG AAC TTT GAC AGA ACC T-3′) or negative control shRNA (LV-NC, 5′-TTC TCC GAA CGT GTC ACG T-3′) were produced using the pGCSIL-GFP vector by Shanghai GeneChem. Recombinant CXCL10 (murine) was purchased from PeproTech (Rocky Hills, NJ, USA). AKT inhibitor IV was purchased from Sigma-Aldrich (St Louis, MO, USA).
For intra-TG microinjection, the mice were deeply anesthetized with isoflurane. The head was stabilized with one hand, and the needle was positioned at an approximately 10° angle relative to the midline of the head and was inserted medial (1–2 mm) to the palpated portion of the zygomatic process through the infraorbital foramen, infraorbital canal, and foramen rotundum and reached the medial wall of the fossa where the TG is located.28 (link) LV-Cxcr3 shRNA (2 µL), CXCL10 (100 ng in 5 µL), or AKT inhibitor IV (0.2 µg in 5 µL) was slowly injected.
For intra-TG microinjection, the mice were deeply anesthetized with isoflurane. The head was stabilized with one hand, and the needle was positioned at an approximately 10° angle relative to the midline of the head and was inserted medial (1–2 mm) to the palpated portion of the zygomatic process through the infraorbital foramen, infraorbital canal, and foramen rotundum and reached the medial wall of the fossa where the TG is located.28 (link) LV-Cxcr3 shRNA (2 µL), CXCL10 (100 ng in 5 µL), or AKT inhibitor IV (0.2 µg in 5 µL) was slowly injected.
4
Optimization of Compound Reconstitution and Dosing
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The following chemicals were obtained from commercial sources, and were used as follows:
(i) The Wnt agonist, AMBMP hydrochloride (Tocris), used as positive control for β-catenin activation, was reconstituted into 10 mM of final concentration in 2 µl DMSO which did not exhibit any cell toxicity (not shown). (ii) AKT Inhibitor IV (Sigma) was used at the concentration of 10 μM in DMSO. (iii) For 250 nM of amlexanox (Tocris), 0.12 µl of 1 mM of stock solution (1 mg/3.35 ml DMSO) was used in 500 µl cell culture. (iv) L-tryptophan (Sigma) was reconstituted at final concentration of 0.50 mM in H2O. (v) Human IFN-γ (Sigma, 1000 units/ml) was used in 1 ml of cell culture. (vi) L-kynurenine (Sigma) was reconstituted at a final concentration of 0.50 mM in H2O. (vii) NAC (Sigma) was made by dissolving in H2O for a final concentration of 5 µM. (viii) For 200 µM of melatonin (N-Acetyl-5-methoxytryptamine, Sigma), 10 mg was dissolved in 430 µl ethanol, and 20 µl was used for 1 ml culture. (ix) From 10 mM of tBHP (Sigma, 1 mg/1.11 ml H2O), 0.1 µl (1 µM) was used for 1 ml culture. (x) For 500 µM of teriflunomide (Tocris), 50 µl of the stock solution in DMSO was used in 1 ml cell culture.
(i) The Wnt agonist, AMBMP hydrochloride (Tocris), used as positive control for β-catenin activation, was reconstituted into 10 mM of final concentration in 2 µl DMSO which did not exhibit any cell toxicity (not shown). (ii) AKT Inhibitor IV (Sigma) was used at the concentration of 10 μM in DMSO. (iii) For 250 nM of amlexanox (Tocris), 0.12 µl of 1 mM of stock solution (1 mg/3.35 ml DMSO) was used in 500 µl cell culture. (iv) L-tryptophan (Sigma) was reconstituted at final concentration of 0.50 mM in H2O. (v) Human IFN-γ (Sigma, 1000 units/ml) was used in 1 ml of cell culture. (vi) L-kynurenine (Sigma) was reconstituted at a final concentration of 0.50 mM in H2O. (vii) NAC (Sigma) was made by dissolving in H2O for a final concentration of 5 µM. (viii) For 200 µM of melatonin (N-Acetyl-5-methoxytryptamine, Sigma), 10 mg was dissolved in 430 µl ethanol, and 20 µl was used for 1 ml culture. (ix) From 10 mM of tBHP (Sigma, 1 mg/1.11 ml H2O), 0.1 µl (1 µM) was used for 1 ml culture. (x) For 500 µM of teriflunomide (Tocris), 50 µl of the stock solution in DMSO was used in 1 ml cell culture.
5
Antibody Sources and Reagents for Lipid Metabolism Research
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Commercial antibodies used are listed in Supplementary Table S2 . Antibody against SCD-1 [57 (link)] was a kind gift from Dr. Jean-Baptiste Demoulin, Université Catholique de Louvain, Brussels, Belgium. Anti-human ACSL4 was generously provided by Dr. Stephen Prescott, University of Utah, Salt Lake, USA and Dr. Diana Stafforini, Huntsman Cancer Institute, University of Utah, USA, and used as indicated in [58 (link)]. Triacsin C was purchased from Santa Cruz (Santa Cruz, CA, USA); A939572 was from Biofine International (Biofine International Inc, Vancouver, Canada); Metformin, Phenformin, AICAR, NS-398 and Akt Inhibitor IV were from Sigma-Aldrich, (Sigma-Aldrich, St. Louis, MO, USA).
6
Differentiation and DENV Infection of MEG-01 Cells
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MEG-01 cells, a human megakaryoblastic leukemia cell line, were grown in RPMI medium (Lonza) with 10% FBS and antibiotic solution (Himedia Laboratories) with 5% CO2. For further differentiation, MEG-01 cells were treated with growth factor TPO (100 ng/mL; PeproTech) for 24 h prior to experiments. For DENV infection, MEG-01 cells were incubated with DENV for 3 h at 37°C, washed with PBS, and resuspended in RPMI-1640 medium with 10% FBS/1X antibiotics and kept at 37°C with 5% CO2 in a cell culture incubator for different experiments. To inhibit different cellular mechanisms, AKT, PI3K, PKCα, and mTOR inhibitors (AKT inhibitor IV (5.0 µM), Ly294002 (10.0 µM), and HBBDE (10.0 µM) from Sigma Aldrich; Torin1 from Cell Signaling Technology) were used respectively as per-requisite of experiments after infection.
7
Neuroprotective Effects of Natural Compounds
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 4′,6-diamidino-2-phenylindole (DAPI), penicillin–streptomycin, and 0.25% (w/v) trypsin containing 1 mM ethylenediaminetetraacetic acid were purchased from Invitrogen (Carlsbad, CA), and 6-OHDA, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), vitamin C, dimethyl sulfoxide (DMSO), Akt inhibitor IV, MEK inhibitor (PD 98059), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Nerve growth factor (NGF) was obtained from R&D Systems (Minneapolis, MN, USA). A total antioxidant capacity assay kit was purchased from Abcam (Cambridge, UK). A lactate dehydrogenase (LDH) cytotoxicity assay kit was purchased from Cayman Chemical (Ann Arbor, MI). A Caspase 3/7 activity detection kit was obtained from Promega (Madison, USA). Antibodies for western blotting were purchased from Cell Signaling Technology (Danvers, MA). All chemicals were dissolved in appropriate solvents and stored at −20°C before use to maintain their chemical stability.
8
Mast Cell Activation Assay
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Dinitrophenyl-human serum albumin (DNP-HSA), anti-DNP-IgE, o-phthaldialdehyde, and 4-nitrophenyl N-acetyl-β-d -glucosamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). The protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from Roche (Mannheim, Germany). IL-3 and stem cell factor (SCF) were purchased from PeproTech (EC, London, UK). The signaling inhibitors, PP2 and Akt inhibitor IV were purchased from Sigma-Aldrich, and U 73122 and LY 294002 (ab120243) were from Abcam (Cambridge, UK). The antibodies for anti-IRF3 and p-S396-IRF3 were obtained from Cell Signaling (Beverly, MA, USA), lamin B1 was obtained from Santa-Cruz Biotechnology (Dallas, TX, USA), and anti-HDC was purchased from Invitrogen (Waltham, MA, USA).
9
Regulation of AKT Signaling in hMSCs
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To regulate the activation of the AKT signaling pathway, 0.01 or 0.1 μM AKT Inhibitor IV (EMD Millipore) was used in hMSC culture. Specifically, ET1-treated hMSCs were cultured with or without AKT Inhibitor IV for two passages before qRT-PCR analysis or induction for multilineage differentiation.
10
Integrin and Cell Signaling Inhibition
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The antibodies, anti-human IgG (Fc) (BioConcept AG, Switzerland), AIIB2 (integrin β1-subunit blocking antibody), P5H9 (αvβ5-integrin blocking antibody) and P1B5 (integrin α3-subunit blocking antibody; all 10 μg/mL supernatants; DSHB, Iowa) were incubated with cells on ice for 30 minutes prior to SCFS. To inhibit cell signaling, wortmannin (100 nM; Sigma-Aldrich)47 (link), LY294002 (50 μM; Cell Signaling Technology)48 (link), NSC23766 (50 μM; Merck Millipore)49 (link), EHT 1864 (100 μM; R&D Systems)50 (link), GGTI 2147 or GGTI286 (10 μM; Merck Millipore)51 (link), Akt inhibitor IV (1 μM; Merck Millipore)52 (link), Akt inhibitor VIII (20 μM; Merck Millipore)53 (link), SecinH3 (20 μM; Merck Millipore)54 (link), blebbistatin (10 μM; Merck Millipore)55 (link) or Y-27632 (10 μM; Merck Millipore)57 (link) were added to the measurement media at the concentrations given. Cells were then incubated for 30 minutes at 37°C prior to SCFS. All inhibitors were dissolved in DMSO and stored at −20°C.
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