Recombinant proteins and general reagents
Protein kinase assays
Cloning and recombinant protein purification from E. coli
Human cell culture and cell treatments
Human cell lysis, immunoprecipitation, and Western blot analysis
Detection of sulfenylated and glutathionylated proteins by immunoblotting
Aurora A sample preparation for MS analysis of intramolecular disulfide bond formation
Liquid chromatography mass spectrometry (LC-MS) analysis of WT and S278C Aurora A
MS data analysis
Identification, alignment, and visualization of protein kinase–related sequences
Yeast strains, plasmids, and growth conditions
Analysis of S. pombe proteins by immunoblotting
Analysis of S. pombe cell length at division and CDC25-GFP localization
Assessing growth and salt stress sensitivity of S. pombe
Statistical analysis
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.