Comparative DSF and peptide-based kinase assay of EphA3 extended R-spine mutants in the presence of various tyrosine kinase inhibitors.

<p><b>A)</b> Analysis of EphA3 WT and extended R-spine mutant proteins. 5 μg or 1 μg of the indicated purified EphA3 proteins were stained with Commassie Blue (top panel) or immunoblotted with a phosphotyrosine antibody (4G10, bottom panel). Increased electrophoretic mobility and lack of autophosphorylated tyrosine are evident in kinase dead (K653M) EphA3 when compared to the other EphA3 proteins analysed. <b>B)</b> Changes in thermal stability in WT and F871A EphA3 measured upon 10μM inhibitor binding. Of the panel of 11 type I and type II kinase inhibitors tested, all induce similar effects on thermostability in both WT EphA3 and the F871A mutant, with the notable exception of dasatinib. WT EphA3 is stabilized to a much greater extent in the presence of dasatinib than the F871A mutant.<b>C)</b> Effects of inhibitors at the indicated concentrations (nM) on WT EphA3 and F871A-catalyzed peptide tyrosine phosphorylation. Activity is normalized to the respective DMSO control with no added inhibitors. The efficacy of inhibition by various inhibitors directly mirrors trends in the thermostability changes induced by inhibitor binding observed in B). <b>D, E)</b> Thermostability effects of dasatinb (D) and ponatinib (E) on full panel of extended R-spine EphA3 mutants. The stabilization effects exhibited by dasatinib differ to those for ponatinib, and this trend is observed for all extended R-spine mutants. Mean ΔTm values ± SD from duplicate experiments were calculated by subtracting the control Tm value (DMSO, no inhibitor (<a href="" target="_blank">S12 Fig</a>)) from the Tm value measured in the presence of inhibitor. * p value <0.05, *** p value <0.001 (Student’s T test).</p>