Figure 6. InsP₆ and InsS₆ inhibit InsP₈ dephosphorylation by PPIP5K2^KD; a luciferase-based ATP assay.

A total of 0.3 μg/ml PPIP5K2^KD was incubated at 37°C with 1.4× reaction buffer (ATP Determination Kit, Molecular Probes), 650 μM ADP and 250 nM InsP₈ for 15 min as described in the Materials and methods section. InsS₆ and InsP₆ were included in the assays as indicated. Then 16 μl of either an ATP standard solution or reaction solution was added into 100 μl Standard Reaction Solution (ATP Determination Kit, Molecular Probes) and luminescence was recorded after 15 s using a BioTek Synergy 2. Data are presented as the means±S.E.M. (n≥3). A dose–response curve was fit using GraphPad Prism v5.03. The IC₅₀ values for InsS₆ and InsP₆ were 0.48 and 0.32 μM, respectively.