Biochemical Tyrosine Kinase Assays: IC50 values for Sunitinib against VEGFR2 (Flk-1) and PDGFRβ are determined using glutathione S-transferase fusion proteins containing the complete cytoplasmic domain of the RTK. Biochemical tyrosine kinase assays to quantitate the trans-phosphorylation activity of VEGFR2 (Flk-1) and PDGFRβ are performed in 96-well microtiter plates precoated (20 μg/well in PBS; incubated overnight at 4 °C) with the peptide substrate poly-Glu,Tyr (4:1). Excess protein binding sites are blocked with the addition of 1-5% (w/v) BSA in PBS. Purified GST-fusion proteins are produced in baculovirus-infected insect cells. GST-VEGFR2 and GST-PDGFRβ are then added to the microtiter wells in 2 × concentration kinase dilution buffer consisting of 100 mM HEPES, 50 mM NaCl, 40 μM NaVO4, and 0.02% (w/v) BSA. The final enzyme concentration for GST-VEGFR2 or GST-PDGFRβ is 50 ng/mL. Twenty-five μL of diluted Sunitinib are subsequently added to each reaction well to produce a range of inhibitor concentrations appropriate for each enzyme. The kinase reaction is initiated by the addition of different concentrations of ATP in a solution of MnCl2 so that the final ATP concentrations spanned the Km for the enzyme, and the final concentration of MnCl2 is 10 mM. The plates are incubated for 5-15 minutes at room temperature before stopping the reaction with the addition of EDTA. The plates are then washed three times with TBST. Rabbit polyclonal antiphosphotyrosine antisera are added to the wells at a 1:10,000 dilution in TBST containing 0.5% (w/v) BSA, 0.025% (w/v) nonfat dry milk, and 100 μM NaVO4 and incubated for 1 hour at 37 °C. The plates are then washed three times with TBST, followed by the addition of goat antirabbit antisera conjugated with horseradish peroxidase (1:10,000 dilution in TBST). The plates are incubated for 1 hour at 37 °C and then washed three times with TBST.The amount of phosphotyrosine in each well is quantitated after the addition of 2,2′-azino-di-[3-ethylbenzthiazoline sulfonate] as substrate.
Cells are starved overnight in medium containing 0.1% FBS prior to addition of Sunitinib and FL (50 ng/mL; FLT3-WT cells only). Proliferation is measured after 48 hours of culture using the Alamar Blue assay or trypan blue cell viability assays. Apoptosis is measured 24 hours after Sunitinib addition by Western blotting to detect cleavage of poly (ADP-ribose) polymerase (PARP) or levels of caspase-3. (Only for Reference)
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