Fig. 1: Eltrombopag directly binds BAK and induces BAK activation.

a Structure of Eltrombopag (Eltro). b After recombinant BAKΔTM was labeled at cysteine using maleimide, microscale thermophoresis (MST) analysis was performed using a series of Eltro concentrations followed by plotting the fraction bound as a function of Eltro concentration. Inset, calculated dissociation constant based on MST assay. c, d After BAK and the indicated concentrations of Eltro or DMSO (negative control) were incubated in CHAPS buffer for 1 h at 25 °C, the mixture was applied to a Superdex75 size exclusion column on an FPLC, and absorbance at 280 nm was plotted as a function of elution volume. A representative experiment (c) and summary from three independent experiments (d) are shown. Labels at top of c indicate size markers. e, f After FITC-dextran-containing lisposome were incubated with the indicated concentration of BAK for 90 min at 25 °C, the percentage of FITC-dextran release was measured. A representative experiment (e) and summary from three independent experiments (f) are shown. g–j After mitochondria from WT (g), Bak^−/− (h), Bax^−/− (i), or Bak^−/−Bax^−/− (j) MEFs were incubated with the indicated concentrations of Eltro or BID BH3 peptide (positive control) at 25 °C for 90 min, the supernatants and pellets were subjected to SDS-PAGE and immunoblotting. A representative result from three independent blotting experiments is shown in g–j.