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
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
Total cell lysates were centrifuged at 20,817g for 20 min at 4°C, and the supernatant was preserved for further analysis. Samples were initially diluted 50-fold, and protein concentration was measured using the Coomassie Plus Staining Reagent (Bradford) Assay Kit (Thermo Fisher Scientific) and processed for immunoblotting with TACC3 or Aurora A antibodies as described (74, 90). To assay the kinase activity of T-loop +2 Cys–containing kinases immunoprecipitated from human cells, WT and Cys-Ala full-length variants of MAPKAP-K2 and PLK1 (C244A and C212A, respectively) were cloned into a pcDNA3 vector and expressed with a 3C protease cleavable, N-terminal Myc tag. All immunoprecipitation experiments used HEK 293T cells transfected using a 3:1 polyethylenimine [average Mw (weight-average molecular weight), ~25,000 Da; Sigma-Aldrich] to DNA ratio (60:20 μg, for a single 10-cm culture dish). Cells were treated with 4 mM valproic acid 24 hours after transfection, and proteins were harvested the following day using a lysis buffer containing 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (v/v) Triton X-100, and 5% (v/v) glycerol and supplemented with a protease inhibitor cocktail tablet and a phosphatase inhibitor tablet (Roche). Lysates were briefly sonicated on ice and clarified by centrifuged at 20,817g for 20 min at 4°C, and the resulting supernatants were incubated with Pierce Anti-c-Myc-Agarose resin (Thermo Fisher Scientific) for 1 hour with gentle end over end mixing at 4°C. Agarose beads containing bound protein were collected and washed three times in 50 mM tris-HCl (pH 7.4) and 500 mM NaCl and then equilibrated in storage buffer [50 mM tris-HCl (pH 7.4), 100 mM NaCl, and 5% (v/v) glycerol]. The purified kinases were then proteolytically eluted from the beads over a 3-hour period using 3C protease (0.5 μg) at 4°C with gentle agitation and then assayed using real-time microfluidic peptide assay as described above.