A Monolith NT.115 instrument (NanoTemper Technologies GmbH) was used for MST analysis. His-TRIB2 was initially labeled with a NanoTemper labeling kit; the fluorescent red dye NT-647 was coupled via NHS chemistry to the N-terminal His-tag, placing the fluorophore away from the pseudokinase domain. For MST, the reaction was performed in 20 mM bicine (pH 9.0), 100 mM NaCl, 5% glycerol, 0.05% Tween 20, and 2% (v/v) DMSO. Fluorescent TRIB2 (~5 μM) was kept constant in the assay, whereas final afatinib and TAK-285 concentrations were titrated over a 3-nM and 50- to 100-μM range. Near-saturation binding was achieved, allowing for an affinity to be estimated for the reversible interaction between afatinib, TAK-285, and fluorescent TRIB2. NT-647–linked MCL-1 and A-1210477 were used as a positive control.

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