Sorted naïve CD4+CD62L+Foxp3YFPCre T cells (1 × 106/ml) were cultured with plate-bound anti-CD28 (5 μg/ml, Biolegend), anti-CD3 (clone: 145–2C11, 5 μg/ml, Biolegend), recombinant TGF-β1 (5 ng/ml, R&D Systems), with or without IL-4 (10 ng/ml) (Peprotech), anti-IL-4 (clone: 11B11, 10 μg/ml) (Biolegend) or anti-IL-6 mAb (MP5–20F3, 10 μg/ml) (Biolegend), mitogen activated protein kinase (MEK) inhibitor PD98059 (50 μM, Sigma-Aldrich) or P38 inhibitor IV (10 μM, Sigma-Aldrich). After 4 days, the induced Treg cells were analyzed by flow cytometry for Foxp3 expression and intracellular cytokines production and/ re-sorted on the basis of YFP expression.
Mek inhibitor pd98059
Manufactured by Merck Group
Sourced in China, Macao, United States
PD98059 is a selective and potent inhibitor of the mitogen-activated protein kinase (MAPK) kinase (MEK) 1 and MEK2 enzymes. It functions by blocking the activation of these enzymes, which are important components of the MAPK signaling pathway. PD98059 is commonly used as a research tool to study the role of the MAPK pathway in various biological processes.
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Lab products found in correlation
P38 inhibitor 4, by Merck Group (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Recombinant tgf β1, by R&D Systems (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Anti il 4 clone, by BioLegend (2 mentions)
Anti il 6 mab mp5 20f3, by BioLegend (2 mentions)
Anti cd28, by BioLegend (2 mentions)
P fak tyr397, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Proteintech (1 mentions)
P plcγ1 tyr783, by Cell Signaling Technology (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Goat anti mouse igg, by Proteintech (1 mentions)
Ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Nerve growth factor (ngf), by R&D Systems (1 mentions)
Akt inhibitor 4, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
2 2 diphenyl 1 picryl hydrazyl hydrate, by Merck Group (1 mentions)
5 protocols using mek inhibitor pd98059
1
Induction and Characterization of Induced T-regulatory Cells
2
Signaling Pathway Investigation Protocol
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Antibodies against PLCγ1, p-PLCγ1 (Tyr783), Caspase-3, AMPKα, p-AMPKα (Thr172), ERK1/2, p-ERK1/2 (Thr202/Tyr204), mTOR, p-mTOR (Ser2448), p-ULK1 (Ser757), FAK, and p-FAK (Tyr397) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Antibodies against P62, Bcl-2, and ULK1 were purchased from Abcam Inc. (Cambridge, MA, USA). Antibody against LC3B was purchased from Novus Biologicals, Inc. (Littleton, CO, USA). Goat anti-Rabbit IgG and Goat anti-Mouse IgG were purchased from Protein Tech Group (Chicago, IL, USA). The dilution concentrations of anti-Rabbit and anti-Mouse were 1:50000 and others were 1:1000. PLCγ1 inhibitor U73122 (U), autophagy inhibitor PI3K inhibitor 3-Methyladenine (3-MA) and Chloroquine (CQ), MEK inhibitor PD98059, mTOR inhibitor Rapamycin, and AMPKα activator Metformin were purchased from Sigma-Aldrich in China (Shanghai, China). Caspase inhibitor Z-VAD-FMK (Z-VAD) and RIP1 inhibitor Necrostatin-1(Nec-1) were purchased from MedChemExpress USA (Monmouth Junction, NJ, USA). Antibodies against Ki67 and MMP2 were purchased from Ruiying Biological (Suzhou, China) and ABclonal (Wuhan,China) respectively. Other reagents were of the highest grade commercially available.
3
Induction and Characterization of Induced T-regulatory Cells
4
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.
5
Dovitinib and BMP-2 Signaling Pathway
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Dovitinib was purchased from Selleck Chemicals (USA) and recombinant human bone morphogenetic protein (BMP)-2 was purchased from R&D systems, Inc. (USA). All cell culture materials including fetal bovine serum (FBS), DMEM, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) were purchased from HyClone (UK). Antibodies against p-ERK1/2, ERK1/2, p-p38, p38, and p-Smad1/5/8 were purchased from Cell Signaling Technology, Inc. (MA, USA). An antibody against actin-horse radish peroxidase (HRP) and Smad1/5/8 were purchased from Santa Cruz Biotechnology, Inc. (USA). The ALP staining kit, MEK inhibitor PD98059, and p38 inhibitor SB202190 were purchased from Sigma Aldrich (USA).
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