Mobility shift assay

Phosphatidylinositol 3‐kinase (p110α/p85α) activity was measured by mobility shift assay (Carna Biosciences, Kobe, Japan). Compound solutions (1000 nM phosphatidylinositol 4,5‐bisphosphate, 50 μM ATP, 5 mM MgCl, and 21 nM PI3K [p110α/p85α]) were prepared with assay buffer containing 2 mM DTT and incubated in a 384‐well microplate for 5 h. The reaction mixtures were then applied to the LabChip (Caliper Life Sciences, Hopkinton, MA, USA), and the product and substrate peaks were quantified. The PI3K reaction was evaluated by the product ratio calculated from peak heights of the product and substrate. The other 312 cellular kinase activities were also evaluated using non‐radioisotopic methods such as the mobility shift assay, ELISA, or IMAP (Carna Biosciences).19 A kinase inhibition profiling panel was produced based on the results of 313 kinase inhibition rates. Histone deacetylase 1 inhibitory activities were evaluated by the Screening Committee of Anticancer Drugs (Tokyo, Japan).

PI3K (p110α/p85α) activity and other 20 common cellular kinase activities were evaluated by the mobility shift assay (Carna Biosciences, Kobe, Japan).19 (link) Individual activity for the four p110 isoforms, α, β, γ and δ, was measured using an homogenous time-resolved fluorescence (HTRF) assay (PI3K assay kit; Millipore). Mammalian target of rapamaycin (mTOR) activity was measured using a K-LISA mTOR activity kit (Millipore). The detailed methods of each assay are described in Supplementary Document S1.