Extended Data Fig. 10: Further characterization of Zn2+ binding in cells.

a, CETSA data related to Fig. 5e. BEAS-2B cells stably expressing FLAG-AKT1(E17K or E17K/C296A/C310A) were treated for 3 h with compound 3 (2 µM) and DMSO or TPEN (10 µM). Thermal stability of FLAG-AKT1 was determined by CETSA with three biological replicates. After treatment with compounds, cells were heat-challenged at the indicated temperatures for 3 min and lysed. Levels of soluble FLAG-AKT1 were determined by dot blot (Biological replicates 1 and 2: plotted as mean ± s.d., three technical replicates. Biological replicate 3: plotted as mean ± s.d., two technical replicates). Apparently missing errors bars indicate that the error is too small to be visualized. Melt temperatures (T_m) were determined by sigmoidal regression analysis. b, BEAS-2B cells stably expressing FLAG-AKT1(E17K) or AKT1(E17K/C296A/C310A) were treated with DMSO or 2 µM compound 3 for 2 h (n = 4). The cells were washed with PBS, lysed, and incubated with anti-FLAG agarose beads for 1 h at 4 °C. The agarose resin was washed 6 times and then FLAG-AKT1 was eluted from the beads with 3xFLAG peptide. Samples were incubated with Chelex 100 resin (15 min), filtered, and concentrated to 57 µL by spin filtration. Samples were analysed by SDS-PAGE with Flamingo dye staining and in-gel fluorescence. Equal volumes of the immunopurified FLAG-AKT1 samples and dilutions of purified recombinant AKT1(E17K) were loaded on the gel. The extrapolated concentrations of immunopurified and eluted FLAG-AKT1 were used to estimate the molar equivalents of transition metals quantified by ICP-MS analysis (Fig. 5f).