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3 protocols using anti phospho s6 2211

1

Lymphocyte Isolation and Lipid Mediator Analysis

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Lymphocyte separation medium, aprotinin, dimethyl sulfoxide (DMSO) and solvents for HPLC and LC/MS were purchased from Thermo Fisher Scientific (Ottawa, Ontario, Canada). Dextran, adenosine deaminase, leupeptin and potassium phosphate were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). HBSS was purchased from Wisent Bioproducts (St-Bruno, Quebec, Canada). 19-OH-prostaglandin (PG) B2, PGB2, PGB2-D4 and 17-octadecynoic acid (17-ODYA) were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). PF-4708671 was obtained from Abcam (Cambridge, Massachusetts, USA) and LY2584702 from Selleckchem (Houston, Texas, USA). Thapsigargin was obtained from Tocris Bioscience (Ellisville. Missouri, USA). LTB4 was a generous gift from Dr Louis Flamand (Université Laval, Québec City, Canada). Recombinant CYP4F3A and the NADPH regenerating system were purchased from Corning (Corning, New York, USA). Protease and phosphatase inhibitor cocktail tablets were purchased from Roche (Laval, Quebec, Canada). Primary (anti-phospho-S6 #2211 and anti-S6 #2317) and secondary antibodies were obtained from Cell Signaling (Danvers, Massachusetts, USA). The enhanced chemiluminescent (ECL) substrate was obtained from Millipore Canada Ltd (Toronto, Ontario, Canada).
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2

Neutrophil Signaling Pathway Activation

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Pre-warmed neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl2) were stimulated with 100 nM of thapsigargin or N-Formylmethionine-leucyl-phenylalanine (fMLP) for 5 minutes. PF-4708671, LY2584702 or vehicle were added 5 minutes before stimulation. Incubations were stopped using 1 volume of cold (4°C) incubation buffer. The suspensions were centrifuged (350 x g, 5 min, 4°C) and then lysed in a cold (4°C) hypotonic buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, pH 7.4) containing 0.1% NP-40, protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupetin, 1 mM PMSF, protease inhibitor cocktail tablets), 2 mM diisopropyl fluorophosphate (DFP) and phosSTOP. Cells were vortexed for 15 seconds, then immediately solubilized in electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol, 2% SDS) and boiled for 10 minutes. Proteins were loaded on a 12% polyacrylamide gel for electrophoresis, and transferred onto a PVDF membrane. Membranes were blocked using TBS/Tween buffer containing 5% w/v skim milk and incubated overnight at 4°C with primary antibodies (anti-phospho-S6 #2211 and anti-S6 #2317, Cell Signaling) in TBS/Tween containing 5% skim milk. HRP-linked secondary antibodies and ECL substrate were used for detection.
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3

FLT3 Mutant Isoform Signaling Analysis

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Exponentially growing Ba/F3 cells stably expressing FLT3 mutant isoforms were plated in RPMI medium 1640 + 10% FCS supplemented with kinase inhibitor at the indicated concentration. After a 90-minute incubation, the cells were washed in phosphate buffered saline (PBS) and lysed in buffer (50mM HEPES, pH 7.4, 10% glycerol, 150mM NaCl, 1% Triton X-100, 1mM EDTA, 1mM EGTA, 1.5mM MgCl2) supplemented with protease and phosphatase inhibitors. The lysate was clarified by centrifugation and quantitated by BCA assay (Thermo Scientific; Rockford, IL). Protein was subjected to sodium dodecyl sulfate polyacrylamide electrophoresis and transferred to nitrocellulose membranes. Immunoblotting was performed using anti-phospho-FLT3 (3464), anti-phospho-STAT5 (9351), anti-STAT5 (9363), anti-phospho-S6 (2211), anti-S6 (2317), anti-phospho-ERK (9101), anti-ERK (9102) (Cell Signaling; Beverly, MA), and anti-FLT3 S18 antibody (Santa Cruz Biotechnology; Santa Cruz, CA). Data shown in figures is representative of 3 technical replicates.
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