Adp hunter plus assay

The kinase activity of PI3K alpha was measured by using the commercial ADP Hunter™ Plus assay (DiscoveRx), a homogeneous assay measuring ADP accumulation, as a universal product of kinase activity. The assay was done following general manufacturer recommendations and adapting protein and substrate concentrations to optimal conditions. Kinase buffer was 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EGTA, 0.04% CHAPS, 3 mM MgCl_2, and 10 μg/ml BGG (bovine γ‐globulin), 2 mM TCEP. The assay was done at 100 μM PIP2, as peptide substrate, and 50 μM ATP. Protein concentration was 0.8 ng/μl. In order to calculate the IC₅₀ of described compounds, serial 1:5 dilutions were prepared, and the reaction started by the addition of ATP. Incubation was done for 1 h at 25°C. Reagents A and B (DiscoveRx) were sequentially added to the wells, and plates were incubated for 30 min at 37°C. Fluorescence counts were read in a Victor instrument (PerkinElmer) with the recommended settings (544 and 580 nm as excitation and emission wavelengths, respectively). Values were plotted against inhibitor concentration and fit to a sigmoid dose–response curve with the GraphPad software.

The PIM kinase activities were measured using the commercial ADP Hunter™ Plus assay (DiscoveRx), a homogeneous assay measuring ADP accumulation, as a universal product of kinase activity. The assay was done following general manufacturer recommendations and adapting protein and substrate concentrations to optimal conditions. Kinase buffer was 15 mM HEPES, pH 7.4, 20 mM NaCl, 1 mM EGTA, 0.02% Tween‐20, 10 mM MgCl_2, and 0.1 mg/ml LBGG (bovine γ‐globulin). All PIM kinase assays were done at 100 μM PIMtide (ARKRRRHPSGPPTA), as peptide substrate, and 100 μM ATP. Protein concentration was 50, 350, and 500 ng/μl for PIM1, PIM2, and PIM3, respectively. In order to calculate the IC₅₀ of described compounds, serial 1:5 dilutions were prepared, and the reaction started by the addition of ATP. Incubation was done for 1 h at 25°C. Reagents A and B (DiscoveRx) were sequentially added to the wells, and plates were incubated for 30 min at 37°C. Fluorescence counts were read in a Victor instrument (PerkinElmer) with the recommended settings (544 and 580 nm as excitation and emission wavelengths, respectively). Values were plotted against inhibitor concentration and fit to a sigmoid dose–response curve with the GraphPad software.