The Invitrogen Zenon Alexa Fluor labeling kit was used for ex pression levels provided in Additional file 1 Table S1. Immunoblotting Cell pellets were lysed with 100 to 150 ul of protein lysis buffer. Pro tein from cell lysates was used for selleck chem ARQ197 whole cell protein analysis after denaturing by Western immunoblot assays using a BioRad Criterion system. Nonspecific binding was blocked by incubating the blots in nonfat dry milk or BSA. Primary antibodies were Inhibitors,Modulators,Libraries incubated for one hour or over night, followed by several washes of Tris buffered saline containing 0. 005% Tween 20. The appropriate secondary antibody was applied for 30, followed by several washes. Antibody re active proteins were detected using a LI COR Odyssey fluorescence optical system.
Immunophenotyping Intracellular AKT protein expression Inhibitors,Modulators,Libraries levels were assayed as follows Cells were fixed and permeabilized using the Fix Perm Fixation and Permeabilization kit. Un labeled primary AKT antibodies were added in a 1 1000 dilution to the cell suspension and incubated for 1 hour at room temperature followed by PBS washing and resuspen sion. Fluorescent dye conjugated secondary antibodies were added in a 1 10 000 dilution and cells were incubated for 30 min at room temperature. After rinsing and resus pension. Site directed mutagenesis and generation of a BaF3 cell line expressing KIT, ABL1 or FLT3 isoforms To compare constitutive activation of AKT mediated by autoactive tyrosine kinase signaling in a homologous cellular background, an isogenic cell model ex pressing different human tyrosine kinase mutations was established.
An IL3 dependent murine pro B cell line was transfected with plasmid Inhibitors,Modulators,Libraries vectors containing cDNA of human FLT3 and KIT isoforms, as well as the BCRABL1 fusion mutation isoform. Gain of func tion tyrosine kinase mutations lead to factor independency. Site directed mutagenesis and generation of a BaF3 cell lines stably expressing mutant KIT D816V, D816Y, Inhibitors,Modulators,Libraries FLT3 ITD, D835V, D835Y, K663Q, BCRABL1 and FLT3 wildtype was previously performed as described before. FLT3 S451F cDNA cloned into a pCMVneo plasmid vector was generously provided by Dr. Fr?hling, University of Ulm, Germany. KIT wildtype cDNA cloned into a pJP1563 plasmid vector was obtained from the DNASU Plasmid Repository at the Biodesign Institute of the Arizona Inhibitors,Modulators,Libraries State University.
Lipofection trans fection into the parental BaF3 cell line was performed to stably express KIT wildtype or mutant FLT3 S451F by double selection for neomycin, blasticidin or gentamicin selleckchem Tofacitinib resistance and IL 3 independent growth. The BaF3 KIT wildtype cell line was cultured using recombinant human stem cell factor as a growth supplement. Apoptosis and proliferation assays Cells were treated in dilution series with the respective small molecule inhibitor.