Thermal shift assay (TSA)

Thermal shift assays were carried out as described in (Niesen et al., 2007). Briefly, in a 96-well RT-PCR plate (Life Technologies) 1 μg HER3 kinase domain/well was incubated with 1 μM inhibitor or 200 μM ATP/10 mM MgCl₂ (as indicated) for 30 mins at 4°C in the presence of Sypro Orange dye (Sigma). HER2 TSA experiments were performed in a 384-well RT-PCR plate (Thermo Fisher Scientific). 0.5 μg of HER2 kinase domain/well was incubated with 1 μM lapatinib, 1 μM bosutinib, or 200 μM ATP/10 mM MgCl₂ for 20 mins at 4°C. HER3 measurements were taken on an Applied Biosystems 7500 Fast Real-Time PCR machine, and HER2 measurements on an Applied Biosystems Quant Studio 7 PCR machine. Data were trimmed and a Boltzmann sigmoidal curve fitted in GraphPad Prism 6. The inflection point of the Boltzmann sigmoidal was taken as the T_m. Thermal shift ΔT_m values were obtained by subtracting the T_m value of the kinase domain alone control.