HEK-293T, MCF7, MDA-MB-231, BT474, T47D, and MDA-MB-468 were maintained in Dulbecco's modified Eagle medium (DMEM; Corning, Manassas, VA, USA), supplemented with 10% fetal bovine serum (FBS; Corning), and Penicillin/Streptomycin (Corning). SUM159PT cells were maintained in Ham's medium (Corning) supplemented with 5% FBS, Hepes (10mM; Sigma-Aldrich, St. Louis, MO, USA), Insulin (5 μg/ml; Sigma-Aldrich), Hydrocortisone (1 μg/ml; Sigma-Aldrich), and Penicillin/Streptomycin. MDA-MB-436 cells were maintained in DMEM:Ham's (1:1) supplemented with 10% FBS, L-Glutamine (2mM; HyClone, Logan, UT, USA), and Penicillin/Streptomycin. Dimethyl sulfoxide (DMSO; Sigma-Aldrich), rapamycin (20 nM; Sigma-Aldrich), U0126 (10 μM; Cell Signaling Technology, Danvers, MA, USA), BI-D1870 (10 μM; Santa Cruz Biotechnology, Dallas, TX, USA), Insulin (100 nM; Sigma-Aldrich), phorbol 12-myristate 13-acetate (PMA) (50 ng/ml; Sigma-Aldrich), etoposide (50 μM; Sigma-Aldrich), and cycloheximide (100 μg/ml; Sigma-Aldrich) were used as indicated in figure legends.
Bi d1870
Manufactured by Santa Cruz Biotechnology
Sourced in United States
BI-D1870 is a small molecule inhibitor that selectively targets the MAP Kinase-Interacting Serine/Threonine-Protein Kinase 1 (MNK1) enzyme. It functions by inhibiting the catalytic activity of MNK1, which plays a role in the regulation of protein synthesis and cell growth.
Automatically generated - may contain errors
Lab products found in correlation
U0126, by Cell Signaling Technology (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Rapamycin, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Etoposide, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Uo126, by Merck Group (1 mentions)
Fr180204, by Merck Group (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Anti g3pdh antibody, by GeneTex (1 mentions)
Anti p csrc antibody, by Cell Signaling Technology (1 mentions)
U0126, by Cayman Chemical (1 mentions)
Anti erk1 2, by Merck Group (1 mentions)
5 protocols using bi d1870
1
Cell Line Maintenance and Treatment
2
Antagonist Assays for NGF Secretion
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Antagonist assays were performed using a 20 minute preincubation of cells with OptiMEM containing the inhibitor. UO126 (Sigma Aldrich), FR180204 (Merck Millipore, Billerica MA USA), BI-D1870 (Santa Cruz Biotechnology, Dallas, TX USA), 217505 (Merck Millipore) were solvated to equal concentrations in DMSO. Literature in vitro IC50 values for intended targets were available as 10–30 nM for BI-D1870 [78] (link) and 72 nM for UO126 [79] (link). Literature in vivo IC50 values for intended targets were available as 2.9 uM for 217505 [80] and FR180204 [81] (link). In expectation that in vitro IC50 values would be lower than in vivo, a range of concentrations was selected from below the predicted IC50 value to above the predicted IC50 value to show dose response as well as complete inhibition. The inhibitor 217505 was poorly soluble above 1 uM under our assay conditions. A complete dose response for 217505 was not performed, as the effects of CREB on NGF gene expression are already well documented and the highest usable dose had no effect on NGF secretion. NGF secretion experiments with UO126, FR180204, and BI-D1870 were always performed in tandem. To facilitate comparisons, NGF secretion experiments with 217505 were vehicle normalized to the other antagonist experiments.
3
Antibody-based Signaling Pathway Analysis
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Antibodies against p-AKT, AKT, cadherin, p-ERK1/2, PARP, p65, p-IκBα, IκBα, p-RSK1, RSK1, p-p27T198 or RhoA and BI-D1870 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-α-tubulin, anti-ERK1/2, and anti-p27 antibody and cycloheximide were purchased from Sigma-Aldrich (St. Louis, MO). Anti-G3PDH antibody was purchased from GeneTex (Hsinchu, Taiwan). Anti-cSrc antibody was purchased from Abcam (Cambridge, MA). Anti-p-cSrc antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA). PP2 was purchased from A. G. Scientific, Inc. (San Diego, CA). U0126 was purchased from Cayman Chemical (Ann Arbor, MI).
4
Cerebellar Slice Preparation and Apoptosis Assay
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The preparation of cerebellar slices followed previously reported methods (Hurtado de Mendoza et al., 2011 (link)). In brief, cerebella were rapidly dissected from P7 to 10 Sct−/− mice and cut into 300-μm slices using a Vibratome (Campden Instruments Ltd., Loughborough, UK) in the iced artificial cerebrospinal fluid (ACSF) containing 2.5 mM calcium chloride and 10 mM D-Glucose. Cerebellar slices were transferred onto 30-mm culture plate inserts with 0.4-μm pores (Millipore, Billerica, MA, USA) in the 6-well plate containing the culture media (75% MEM, 25% heat-inactivated horse serum, 25 mM HEPES, 1 mM glutamine, 5 mg/mL glucose, 100 U/mL penicillin and streptomycin). To investigate the mechanisms underlying SCT-mediated apoptosis, cerebellar slices were incubated with graded concentration of SCT (0, 0.01, 0.1, 1 and 10 μM; AnaSpec Inc., San Jose, CA, USA) for 30 min. Pretreatment with H89 (20 μM; Sigma-Aldrich, St. Louis, MO, USA), and/or U0126 (20 μM; Cell Signaling Technology, Danvers, MA, USA), or BI-D1870 (20 μM; Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min was performed prior to the 30-min incubation with 1 μM SCT. All slices were incubated in a humidified chamber at 37°C with 5% CO2.
5
EphA2 and Akt Signaling Pathway Inhibitors
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The pharmacological MEK inhibitors U0126 and PD98059, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were purchased from Merck Millipore. Akt inhibitor MK-2206 was from ChemieTek, and RSK inhibitor BI-D1870 was from Santa Cruz Biotechnology. We used the following antibodies in this study: a rabbit polyclonal antibody against EphA2 (C-20) (Santa Cruz Biotechnology); a mouse monoclonal antibody against a-tubulin (Sigma); rabbit monoclonal antibodies against S897 phospho-EphA2 (D9A1) and T308 phospho-Akt (C31E5E), rabbit polyclonal antibodies against Akt, ERK, and T202/Y204 phospho-ERK, a mouse monoclonal antibody against EphA2 (8B6) (Cell Signaling Technology); a mouse monoclonal antibody against HA (3F10) (Millipore); secondary antibodies conjugated to horseradish peroxidase (DAKO); Alexa Fluor 488-conjugated anti-GFP and Alexa Fluor 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific).
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