PI4KIIIbeta-IN-10

Kinase experiment:

Lipid kinase assays are preformed using recombinant enzyme, phosphoinositides and γ32P-ATP in a membrane capture assay. Each inhibitor (e.g., PI4KIIIbeta-IN-10) is diluted into 10% DMSO and kinase assay buffer. Upon completion of the reaction, 4 µL is spotted onto 0.2 µm nitrocellulose. The membrane is dried for 5 minutes under a heat lamp followed by 1×30 second wash and 6×5 min washes in 1M NaCl /1% Phosphoric Acid. The membrane is dried for 20 minutes under a heat lamp followed by overnight exposure to a phosphor screen and phosphorimaging followed on a Typhoon 9500. Intensities are quantified using SPOT. Specifications for each enzyme follow. L-α-Phosphatidylinositol and DOPS:DOPC lipids are sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction is set-up as follows 1) kinase assay buffer, PI:DOPS:DOPC, BSA and PI4KIIIβ, are combined in a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution is added (5x solution) and incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ32P-ATP are added (2.5x solution) to initiate the reaction which ran for 30 minutes. Final conditions are as follows: 20 mM Bis-Tris Propane pH 7.5, 10 mM MgCl2, 0.075 mM Triton X-100, 0.5 mM EGTA, 1 mM DTT, 100 µM PI, 500 ng/µL BSA, 2.5 nM PI4KIIIβ, 2% DMSO, 10 µM ATP and 1 uCi γ32P-ATP[1].

References:

[1]. Rutaganira FU, et al. Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ. J Med Chem. 2016 Mar 10;59(5):1830-9.