Thermal shift assays were performed with a StepOnePlus real-time polymerase chain reaction (PCR) machine (Life Technologies) using SYPRO Orange dye (Invitrogen) and thermal ramping (0.3°C in step intervals between 25° and 94°C). All proteins were diluted to a final concentration of 5 μM in 50 mM tris-HCl (pH 7.4) and 100 mM NaCl in the presence or absence of the indicated concentrations of ATP, H2O2, DTT, GSH, or MLN8237 [final dimethyl sulfoxide (DMSO) concentration no higher than 4% (v/v)] (131) and were assayed as described previously (67). Normalized data were processed using the Boltzmann equation to generate sigmoidal denaturation curves, and average Tm/∆Tm values were calculated as previously described (132) using GraphPad Prism software.

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