Kinase Assay: For selectivity profiling experiments, the lysates (5 mg of total protein each) are preincubated with 0 (DMSO control), 2.5 nM, 25 nM, 250 nM, 2.5 μM or 25 μM free compound (GSK690693 or GSK2141795) on an end-over-end shaker for 45 min at 4°C. Subsequently, lysates are incubated with beads (coupled Akt probe or kinobeads) for 1 h at 4°C, for both qualitative and quantitative experiments. The beads are washed with 1×CP buffer and collected by centrifugation. Bound proteins are eluted with 2×NuPAGE LDS sample buffer, and eluates are reduced and alkylated by 50 mM dithiothreitol and 55 mM iodoacetamide.

Cell Assay: Cells were washed in ice-cold tris buffered saline (TBS), spun down and lysed in radioimmunoprecipitation assay buffer (10 mM Tris HCl, pH 8, 5 mM Na2EDTA, pH 8, 1% NP-40, 0.5% sodium dioxycholate, 0.1% SDS), both containing protease and phosphatase inhibitors (Complete Mini PhosphoSTOP, Roche, Basel, Switzerland). Protein concentrations were determined by Pierce BCA Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. 5–40 μg protein was resolved on 4–12% RunBlue SDS-PAGE gels (Expedeon, San Diego, CA, USA), transferred onto PVDF membrane (GE Healthcare Life Sciences, Buckinghamshire, UK), blocked and then incubated with primary monoclonal antibodies ON at 4 °C. Following, the membranes were incubated with goat anti-rabbit, goat anti-mouse (#P0448, #P0447, Dako, Glostrup, Denmark) or donkey anti-goat (#SC-2020, Santa Cruz Biotechnology, Heidelberg, Germany) HRP-conjugated secondary antibodies in 1:5000 dilution for 1 h at room temperature (RT). The immune reactive bands were visualized by Amersham ECL Prime Western Blotting Detecting Reagent (GE Healthcare Life Sciences) and exposed to CL-Xposure film (Thermo Fisher Scientific).