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
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
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
The Multiply-Aligned Profiles for Global Alignment of Protein Sequences (MAPGAPS) procedure (135) was used alongside a variety of curated ePKs profiles (129, 136–138) to identify and align ePK-related sequences from the nonredundant sequence database and UniProt reference proteome (139) databases (Release 2018_09). Sequences with a Cys residue at the Aurora A Cys290-equivalent position were retrieved and used for further taxonomic analysis. Taxonomic information was based on National Center for Biotechnology Information Taxonomy database (140) and WebLogos (141) were generated using WebLogo version 2.8. Amino acids were colored on the basis of their chemical properties. Polar (G, S, T, Y, C, Q, and N), basic (K, R, and H), acidic (D and E), and hydrophobic (A, V, L, I, P, W, and M) amino acids are colored green, blue, red, and black, respectively.