Recombinant enzyme

Recombinant enzyme was purchased from EMD Millipore (Cat # 14–908, Lot # D9KN031N–B). L-α-Phosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat # 790595P) were sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was set-up as follows 1) kinase assay buffer, PI:DOPS:DOPC, BSA and PI4KIIIα, were combined in a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ³²P-ATP were added (2.5x solution) to initiate the reaction which ran for 25 minutes. Final conditions were as follows: 20mM Bis-Tris Propane pH 7.5, 10mM MgCl₂, 0.075mM Triton X-100, 0.5mM EGTA, 1mM DTT, 100µM PI, 500ng/µL BSA, 1.2nM PI4KIIIα, 2% DMSO, 10µM ATP and 2uCi γ³²P-ATP.

Recombinant enzyme was purchased from EMD Millipore (Cat # 14–910, Lot # 2023057-A). L-α-Phosphatidylinositol (PI, Cat # 840024P) and DOPS:DOPC lipids (Cat # 790595P) were sonicated in water to generate 1mg/mL PI:DOPS:DOPC. Reaction was set-up as follows 1) kinase assay buffer, PI:DOPS:DOPC, BSA and PI3KC2γ, were combined in a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ³²P-ATP were added (2.5x solution) to initiate the reaction which ran for 15 minutes. Final conditions were as follows: 50mM HEPES pH 7.5, 100mM NaCl, 0.03% CHAPS, 10mM MgCl₂, 1mM EGTA, 2mM DTT, 1nM PI3KC2γ, 100µM PI, 500ng/ µL BSA, 2% DMSO, 10µM ATP and 2uCi γ³²P-ATP.

Recombinant enzyme was purchased from EMD Millipore (Cat # 14–602, Lot # 2150294-A). L-α-Phosphatidylinositol-4,5-bisphosphate (PIP₂, Cat # 840046P) and DOPS:DOPC lipids (Cat # 790595P) were sonicated in water to generate 1mg/mL PIP₂:DOPS:DOPC. Reaction was set-up as follows 1) kinase assay buffer, PIP₂:DOPS:DOPC, BSA and PI3Kα, were combined in a total volume of 10 µL (2.5x solution); 2) 5 µL of inhibitor solution was added (5x solution) and incubated with enzyme mixture for 15 minutes; 3) 10 µL cold ATP and γ³²P-ATP were added (2.5x solution) to initiate the reaction which ran for 15 minutes. Final conditions were as follows: 50mM HEPES pH 7.5, 100mM NaCl, 0.03% CHAPS, 10mM MgCl₂, 1mM EGTA, 2mM DTT, 3.3nM PI3Kα, 80µM PIP₂, 500ng/ µL BSA, 2% DMSO, 10µM ATP and 2uCi γ³²P-ATP.