Recombinant proteins and general reagents
Protein kinase assays
Cloning and recombinant protein purification from E. coli
Differential scanning fluorimetry
Human cell culture and cell treatments
Human cell lysis, immunoprecipitation, and Western blot analysis
Detection of sulfenylated and glutathionylated proteins by immunoblotting
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
Five micrograms of Aurora A purified in the absence of DTT was heated at 80°C in 0.06% (w/v) RapiGest SF (Waters) dissolved in 25 mM ammonium bicarbonate for 10 min. Samples were digested overnight at 25°C using a 20:1 (w/w) ratio of Aurora A:Chymotrypsin (Promega), with shaking at 600 rpm. The sample was equally split into two, one aliquot of which was reduced with DTT (3 mM) at 60°C for 10 min, cooled, and alkylated with 10 mM iodoacetamide (IAA) at room temperature for 30 min in the dark. Excess IAA was quenched by addition of DTT to a final concentration of 7 mM. The second sample was left on ice. RapiGest hydrolysis was induced in both samples by the addition of 1.5% (v/v) trifluoroacetic acid (TFA) and 3% (v/v) acetonitrile, shaking at 600 rpm and 37°C for 2 hours. Insoluble products were removed by centrifugation (13,000g for 15 min at 4°C). Samples were subjected to strong cation exchange using in-house packed stage tips (Empore Supelco 47-mm Cation Exchange disk #2251), three disks per 200 μl of tip. All centrifugation steps were performed at 2000g for 3 min until all liquid had passed through the stage tip. Briefly, tips were equilibrated with sequential washes in acetone, methanol, water, 5% (v/v) ammonium hydroxide, and water (2 × 200 μl of each). Peptide samples were thrice passed through the equilibrated stage tip and washed with 250 μl of 1.5% (v/v) TFA, before elution with 250 μl of 5% (v/v) ammonium hydroxide. Eluted material was dried using cooled vacuum centrifugation. Peptides were solubilized in 20 μl of 3% (v/v) acetonitrile and 0.1% (v/v) TFA and sonicated for 10 min before centrifugation (13,000g for 15 min at 4°C) to remove insoluble material before LC-MS/MS analysis.