RGC-5 cells were transfected with Nmnat1 siRNA or Nmnat1 plasmid to change Nmnat1 expression levels. After that, RGC-5 cells were pretreated with MAPK inhibitors (U0126, 10 µM; SB203580, 10 µM; SP600125, 10 µM) for 1 h to inhibit MAPK signaling, and then exposed to high glucose (30 mM) for 48 h. U0126 (ERK inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) were purchased from Sigma Chemical.
U0126
Manufactured by Merck Group
Sourced in United States, Germany, France, China, United Kingdom, Sao Tome and Principe, Macao, Canada, Italy, Japan
U0126 is a selective and potent inhibitor of the mitogen-activated protein kinase (MAPK) kinases, MEK1 and MEK2. It blocks the phosphorylation and activation of the extracellular signal-regulated kinases (ERK1 and ERK2), thereby inhibiting the MAPK/ERK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Sb203580, by Merck Group (94 mentions)
Sp600125, by Merck Group (81 mentions)
Ly294002, by Merck Group (80 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (50 mentions)
Pd98059, by Merck Group (40 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (36 mentions)
Wortmannin, by Merck Group (31 mentions)
Sb202190, by Merck Group (22 mentions)
Rapamycin, by Merck Group (19 mentions)
Bay11 7082, by Merck Group (19 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (18 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (16 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (15 mentions)
β actin, by Santa Cruz Biotechnology (13 mentions)
Erk1 2, by Cell Signaling Technology (12 mentions)
Streptomycin, by Thermo Fisher Scientific (12 mentions)
Sb431542, by Merck Group (11 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions)
Penicillin, by Thermo Fisher Scientific (11 mentions)
Ag1478, by Merck Group (10 mentions)
613 protocols using u0126
1
Nmnat1 Expression and MAPK Inhibition in RGC-5 Cells
2
Quantifying Heterotypic Cell Interactions
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Cells were pre-labelled with either CellTracker Red or Green, counted and combined in 1:1 suspension so that 5×104 of each cell type was seeded per well in a 24-well imaging µ-Plate (Ibidi) for a total of 1×105 cells/well. For wild-type assays, cells were allowed 6 h to adhere before being imaged overnight for a total of 24 h. For RNAi experiments, cells were transfected with siRNA for 48 h before being differentially labelled, combined, and allowed to mix for 24 h before fixation with 4% paraformaldehyde in PBS. All nuclei were then labelled with DRAQ5 (Invitrogen) before coverslips were mounted. Images were then acquired, and the green channel was used to create a mask over the nuclei of HT1080 cells, leaving only the nuclei of HaCaT cells visible for segmentation. Nearest neighbour distances were then calculated in ImageJ. For U0126 MAPK/ERK kinase (MEK) inhibition movies, cells were differentially labelled before being combined and allowed to adhere for 6 h. U0126 (Sigma-Aldrich) was then added at a concentration of 20 µM to inhibit MEK/ERK signalling alongside DMSO as vehicle control, and live imaging commenced for a total of 24 h. For quantitative analysis, HaCaT cells at 6 h and 24 h were manually segmented and nearest neighbour distances were calculated in ImageJ.
3
Preadipocyte Response to CaSR Activators
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Isolation of human AT-derived (primary) preadipocytes was performed as described previously [17 (link)]. After passage 3 in culture with DMEM:F12 and 10% FBS, preadipocytes were switched to 2.5% FBS and exposed to cinacalcet, spermine and/or the upstream ERK (MAPK) inhibitor U0126 (#U120, Sigma-Aldrich, St Louis, MO, USA), plus the autophagy flux inhibitor CQ for the last 2–3 h. CaSR activators were used for 6 h, while U0126 was used 1 h before exposure to the CaSR activator.
4
MEK Inhibitor Treatment of Embryos
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Embryos were obtained by in vitro fertilization, then chemically dechorionated and grown in MFSW at room temperature, until the desired stage. Experimental embryos were treated with 4 μM MEK inhibitor compound U0126 (Sigma) in MFSW (U0126 stock solution: 2 mM in dimethylsulphoxide) (Sigma) for 30 min and then washed with MFSW to remove the compound. Treated embryos were allowed to develop to the desired stage and then fixed for whole-mount ISH or immunohistochemistry.
5
Cytotoxicity Screening of Pharmacological Agents
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U0126, AG1024, LY294002, vincristine, and daunorubicin were dissolved in dimethylsulfoxide (DMSO) and dexamethasone in 100% ethanol at stock concentrations of 10mmol/L. Cells were incubated with U0126, AG1024, vincristine, daunorubicin, or dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) at the indicated doses at 37°C for 72hours and subsequently reacted with Cell TitreGlo reagent according to the manufacturer's instructions (Promega). Luminescence was quantified using a Victor Wallac plate reader.
6
Fluorescence Imaging of Cell Signaling
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Before imaging, 35-mm glass-bottom dishes were washed twice gently with phosphate-buffered saline (PBS; Gibco, #70011036) and then imaged in the dark in either calcium-free Hank’s balanced salt solution (HBSS; Gibco, #14185052) supplemented with 0.2% (w/v) bovine serum albumin (BSA; Gibco, #10270106) (HEK293T, COS-7) at room temperature, or in FluoroBrite DMEM supplemented with 0.5% (v/v) FBS and 1× Glutamax (HeLa) at 37 °C, 5% CO2. Cells were treated at indicated timepoints using a final concentration of 50 μM forskolin (Fsk) (Sigma-Aldrich, #93049), 100 μM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, #410957), 1 μg/ml epidermal growth factor (EGF) (R&D Systems, #236-EG10), 10 μM ionomycin (Iono) (Sigma-Aldrich, #407950), 2 mM CaCl2 (Chem-Lab Analytical, #CL05.0371), 1 μM phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, #P1585), 20 μM U0126 (Sigma-Aldrich, #662005) and 20 μM H-89 (Sigma-Aldrich, #B1427), by mixing a 5× concentrated solution, prepared in imaging buffer from freshly thawed DMSO (Fsk, IBMX, PMA, U0126, H-89) or HBSS (CaCl2) stocks, into the imaging dish.
7
Gene Expression Analysis of Cell Signaling Inhibitors
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Cells were seeded onto 24-well plates (5 × 104 cells per well). After 2 h at 37 °C and 5% CO2 to allow for cell adhesion and spreading, cells were incubated with culture media (2% charcoal-stripped FBS) containing either U0126 (30 μM) (Sigma-Aldrich), MK2206 (9 μM) (Sigma-Aldrich), U0126 (30 μM) and MK2206 (9 μM), or vehicle (DMSO) only for 24 h. Total RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Courtaboef, France) as previously described2 (link)8 (link)37 (link)38 (link)39 (link). RNA yield and integrity were analyzed using the RNA 6000 Pico kit and the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) as previously described2 (link)8 (link)37 (link)38 (link)39 (link). mRNA levels of cyclin D1, p53, and p21WAF1/Cip1 (p21) were measured by quantitative real-time RT-PCR with a Light Cycler (Roche, Mannheim, Germany) as previously described2 (link)8 (link)37 (link)38 (link)39 (link). Here, Primer sets are shown in Supplementary Table S4 .
8
Pancreatic Cancer Cells Treated with U0126
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The pancreatic cancer cells were seeded in 100-mm culture dishes at 5 × 105 cells per dish with an appropriate culture medium containing 10% FBS, then cultured under 37 °C in 5% CO2 with the appropriate humidity. After 24 h, the medium was replaced with a medium containing U0126 (Sigma-Aldrich, St. Louis, MO, USA), a MAP2K inhibitor, dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 10 μmol/L, or replaced with a medium containing the same volume of DMSO without U0126 as a control. After 24 h, RNA isolation and protein extraction were carried out.
9
Cell Migration and Invasion Assay
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Migration and invasion assays were performed as described previously [22 (link)]. Briefly, cells were plated onto cell culture inserts with 8μm microporous filters (Corning) coated with (invasion) or without (migration) 40 μL Matrigel (1:8 dilution; BD Biosciences, Bedford, MA) and incubated for 24 h. Cells in the upper filters (inside the inserts) were removed, and cells that had migrated into or invaded the lower filters were fixed in 4% paraformaldehyde, stained with crystal violet and counted under a microscope. The number of migrated or invaded cells was counted in five random optical fields for each filter (100 × magnification). For A549 and L78 cells, 2 × 104 and 4 × 104 cells were plated onto each insert, respectively. To test the effect of the specific MEK1/2 inhibitor U0126 (Sigma) on the migratory and invasive abilities of cells, A549 and L78 cells were pretreated with 10 μM U0126 for 30 min before migration and invasion assays were performed. Experiments were performed in triplicate.
10
BMSC Treatment with 7,8-DHF and Inhibitor
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The compound 7, 8-DHF was purchased from Sigma (⩾98%). 7, 8-DHF was dissolved in PBS with 17% dimethyl sulfoxide (DMSO). BMSCs were seeded into 6-well culture plates at a density of (2 × 107 cells/well) and cultured to ~90% confluence. BMSCs were treated with 7, 8-DHF using an optimal concentration of 500 nMol/L, as determined in a previous study, for 72 h.24 (link)
Fresh culture medium was replaced, and the BMSCs were separated into four experimental groups: (1) a negative control (NC) group, treated with medium only, (2) cells treated with 7, 8-DHF, (3) 7, 8-DHF + U0126 group, the cells were treated with 7, 8-DHF and U0126 (selective inhibitor of ERK1/2 signaling, 5 μM, Sigma-Aldrich, St. Louis, MO, USA), and (4) BDNF group, the cells were treated with human recombinant BDNF (159 nMol/L, Sigma).18 (link)
Following the treatment period, protein was isolated from the BMSCs for ELISA and Western blot analysis.
Fresh culture medium was replaced, and the BMSCs were separated into four experimental groups: (1) a negative control (NC) group, treated with medium only, (2) cells treated with 7, 8-DHF, (3) 7, 8-DHF + U0126 group, the cells were treated with 7, 8-DHF and U0126 (selective inhibitor of ERK1/2 signaling, 5 μM, Sigma-Aldrich, St. Louis, MO, USA), and (4) BDNF group, the cells were treated with human recombinant BDNF (159 nMol/L, Sigma).18 (link)
Following the treatment period, protein was isolated from the BMSCs for ELISA and Western blot analysis.
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