HepG2 cells (10,000 cells/well) in DMEM with 10% FBS were seeded into a 96-well plate and treated with 5 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA), 10 μM SB431542 (Cayman, Ann Arbor, MI, USA), 1 μM MEK1/2 inhibitor U0126 (Tocris, Bristol, UK), or a combination of TGF-β1 and SB431542 or U0126. In the control group, HepG2 cells were treated with vehicle (4 μM HCl, 0.001% BSA, and 0.025% DMSO).
U0126
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
U0126 is a potent, selective, and cell-permeable inhibitor of the mitogen-activated protein kinase (MAPK) kinases MEK1 and MEK2. It effectively blocks the activation of the ERK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Bio-Techne (16 mentions)
Sp600125, by Bio-Techne (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
U73122, by Bio-Techne (6 mentions)
Sb203580, by Bio-Techne (5 mentions)
Wortmannin, by Bio-Techne (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Sb431542, by Bio-Techne (4 mentions)
Sr11302, by Bio-Techne (4 mentions)
Sb202190, by Bio-Techne (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Ag490, by Bio-Techne (3 mentions)
Kn 93, by Bio-Techne (3 mentions)
N acetyl l cysteine, by Merck Group (3 mentions)
Pd98059, by Bio-Techne (3 mentions)
99 protocols using u0126
1
TGF-β1 Signaling Pathway Modulation
2
Evaluating STAT3 and MAPK/ERK1/2 Inhibition in Metastatic Breast Cancer
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Starved metastatic breast cancer cell line, MDA-MB-231, was treated with the STAT3 inhibitor NSC74859 (300 nM; Tocris) or the MAPK/ERK1/2 inhibitor U0126 (20 nM; Tocris) for 24 h in both regular media as well as astrocytic media and assessed using the MTT Cell Growth Assay Kit (Chemicon International Inc., Temecula, CA, USA). A Multiscan FC Microplate Reader (Thermo Fisher Scientific) was utilized to measure absorbance (595 nm and 620 nm). Vehicle treatment was given to controls. Final results are presented as the mean±SEM.
Migration assays. Scratch wound healing assays were used to assess cell migration in the presence of the STAT3 inhibitor NSC74859 (300 nM; Tocris) or the MAPK/ERK1/2 inhibitor U0126 (20 nM; Tocris). Vehicle-treated cells were used as a control. A monolayer of MDA-MB-231 cells was wounded, and cell movement was subsequently monitored over a period of 24 h using the Axiovert 200 microscope (ZEISS, Oberkochen, Germany) at 3.6x magnification. The migration rate was determined by measuring the mean distance between borderlines of the wound. Final results are presented as the mean±SEM.
Statistical analysis. Analysis of variance (ANOVA) tests followed by Student's t-test (unpaired, 2-tailed) were used to evaluate significant variations between control and experimental results. Significance was indicated by a p-value less than 0.05.
Migration assays. Scratch wound healing assays were used to assess cell migration in the presence of the STAT3 inhibitor NSC74859 (300 nM; Tocris) or the MAPK/ERK1/2 inhibitor U0126 (20 nM; Tocris). Vehicle-treated cells were used as a control. A monolayer of MDA-MB-231 cells was wounded, and cell movement was subsequently monitored over a period of 24 h using the Axiovert 200 microscope (ZEISS, Oberkochen, Germany) at 3.6x magnification. The migration rate was determined by measuring the mean distance between borderlines of the wound. Final results are presented as the mean±SEM.
Statistical analysis. Analysis of variance (ANOVA) tests followed by Student's t-test (unpaired, 2-tailed) were used to evaluate significant variations between control and experimental results. Significance was indicated by a p-value less than 0.05.
3
Protein Expression and Localization
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Primary antibodies used in this study are listed the reagent table. Most of the primary antibodies were used for immunofluorescence at the dilution of 1:50 to 1:100 except VEcad (1:400) and α-Tub (1:500). For Western blotting, the primary antibody dilution was 1:500 for all, except Cx37 (1:250). For immunofluorescence, the species-specific secondary antibodies used in this study were conjugated with Alexa Fluor 405, 488, 546, 594, and 647 (1:500; Thermo Fisher Scientific) or with horseradish peroxidase-conjugated secondary antibodies (1:2,500/5,000; Cell Signaling Technologies) for Western blotting.
Cells were treated with DAPT (5 μM for 12 h, Cat#D5942; Sigma-Aldrich), LY294002 (10 μM for 1 h, or 2.5 μM for 12 h; Cat# 1130; Tocris), U0126 (10 μM for 1 h, or 2.5 μM for 12 h; Cat# 9903; Cell Signaling Technology), MK8353 (2.5 μM for 12 h; Cat# SCH900353; SelleckChem), nocodazole (10 μM for 30 min; Cat#M1404; Sigma-Aldrich), and BAPTA-AM (10 μM for 2 h, Cat#A1076; Sigma-Aldrich).
Cells were treated with DAPT (5 μM for 12 h, Cat#D5942; Sigma-Aldrich), LY294002 (10 μM for 1 h, or 2.5 μM for 12 h; Cat# 1130; Tocris), U0126 (10 μM for 1 h, or 2.5 μM for 12 h; Cat# 9903; Cell Signaling Technology), MK8353 (2.5 μM for 12 h; Cat# SCH900353; SelleckChem), nocodazole (10 μM for 30 min; Cat#M1404; Sigma-Aldrich), and BAPTA-AM (10 μM for 2 h, Cat#A1076; Sigma-Aldrich).
4
Fibroblast and HeLa Cell Culture
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Control (Coriell Institute, USA) and JNCL fibroblasts (Gaslini Institute, Italy) were grown in DMEM (1:1, HyClone) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals), 2 mM L -glutamine, 100 U ml−1 penicillin and 100 mg ml−1 streptomycin (Invitrogen). HeLa cells were incubated for 2 h with LY294002 (50 mM, Cell Signaling), Torin 1 (300 nM, Cayman Chemical) or for 24 h with trehalose (100 mM, Sigma), rapamycin (300 nM, Sigma), MK2206 (1 μM, Selleckchem), U0126 (10 μM, Tocris) and dialyzed serum (GE Healthcare Life Sciences) for 30 min.
5
Calpain and AMPA Receptor Inhibitors
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The following drugs were used: MDL28170 a potent, selective inhibitor of calpain (Ki = 10 nM; Tocris, #1146); CNQX an AMPA receptor antagonist (IC50 = 1–2 μM; Tocris, #1045); ω-agatoxin a selective and reversible blocker of Cav2.1 (P/Q-type VGCC) (Alomone, #A-530); K252a a non-selective protein kinase inhibitor that inhibits PKC (IC50 = 32.9 nM; Tocris, #1683); U0126 a selective non-competitive inhibitor of MEK-1 and MEK-2 (IC50 = 60–70 nM; Tocris, #1144).
6
Cell Proliferation and Signaling Assays
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Dulbecco's modified Eagle's medium (DMEM) and FBS were purchased from Gibco‐BRL (by Thermo Fisher Scientific Inc.; Waltham, MA, USA). MG‐132 was a product of Enzo Life Sciences (Exeter, UK). Cycloheximide (CHX) and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). SP600125, U0126, and SB203580 were purchased from Tocris Bioscience (Bristol, UK). Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix was supplied by CORNING Inc. (Corning, NY, USA).
7
Protocols for Neural Stem Cell Culture
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Laminin, poly‐ornithine, thyroid hormone (T3), paraformaldehyde (PFA), bis(cyclohexanone)oxaldihydrazone (Cuprizone), Hoechst 33342, poly‐d ‐lysine, papain, l ‐cysteine, insulin, transferrin, progesterone, putrescine, BSA, and 5‐fluoro‐2′‐deoxyuridine were purchased from Sigma‐Aldrich. Collagenase A was purchased from Roche. EGF, bFGF, and PDGF‐AA were purchased from Peprotech. PD0325901, CI‐1040, AZD8330, and AZD6244 were purchased from MedChemExpress, and U0126 was purchased from Tocris.
8
Reagents for Cell Culture Experiments
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All reagents used were cell culture grade. Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM, Hanks’ balanced salt solution (HBSS), trypsin-EDTA, fetal bovine serum (FBS), penicillin, streptomycin, and dispase were obtained from Invitrogen (Carlsbad, CA). Bovine serum albumin (BSA) was from Equitech-Bio Inc. (Kerrville, TX). Thrombin, hirudin, KN-93, and Ro-32–0432 were from EMD Millipore (Burlington, MA). Ser-Phe-Leu-Leu-Arg-Asn-amide trifluoroacetate salt (SFLLRN), amino-oxyacetic acid (AAOA), L-methionine sulfoximine, EGTA, ryanodine, dantrolene, and wortmannin were obtained from Merck KGaA (Darmstadt, Germany). L-trans-2, 4-L-trans-Pyrrolidine-2,4-dicarboxylic acid (PDC), U73122, U0126, and KB-R7943 were from Tocris Bioscience (Bristol, UK). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA AM) was obtained from Molecular Probes (Eugene, OR). D-Phenylalanyl-prolyl-arginyl Chloromethyl Ketone (PPACK) was from Enzo Life Sciences (New York, NY).
9
Molecular Mechanisms Regulating Cell Signaling
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Dulbecco’s modified Eagles’ medium (DMEM), penicillin/streptomycin, trypsin/EDTA, fetal bovine serum (FBS), KGM-SFM medium, and phosphate-buffered saline (PBS) were from Gibco (Life Technologies, USA). Arecoline, melatonin and MTT were from Sigma (Sigma-Aldrich Chemical Company, St. Louis, MO, USA). ELISA kits for MMP-9 were from PeproTech (PeproTech, Inc., Rocky Hill, NJ, USA). Catalase, SB431542, 5Z-7-Oxozeaenol, PD153035, AG490, U0126, LY294002, and aspirin were purchased from Tocris. Phospho-TAK1 (p-TAK1) antibody was from Abcam (ab79583). Antibodies for TGF-β1, and GAPDH were from Santa Cruz. P-Smad2 antibody was from Cell Signaling Technology. PBL extract and ANE were prepared as before [8 (link), 12 (link), 13 (link), 21 (link)]. HC was synthesized as previously [22 (link)].
10
Inhibitor Screening for Cell Signaling
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U0126 (Cat. # 1144, MEK inhibitor) and Go6976 (Cat. # 2253/1, PKCα inhibitor) were purchased from TOCRIS (Bristol UK). TBCA (Cat. # 934358–00–6, CK2 inhibitor) and S2101 (Cat. # 48977, LSD1 inhibitor) were purchased from Calbiochem (San Diego, CA, USA). SCH772984 (Cat. # CT-SCH772, ERK inhibitor) and Tamoxifen were purchased from Chemie Tek (Indianapolis, IN, USA) and Sigma, respectively. Dual-Luciferase Reporter Assay system was purchased from Promega (Madison, WI, USA).
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