Kinase assays were performed using nonradioactive real-time mobility shift-based microfluidic assays, as described previously (66, 91, 129, 130), in the presence of 2 μM of the appropriate fluorescent-tagged peptide substrate (table S2) and 1 mM ATP (unless specified otherwise). Pressure and voltage settings were adjusted manually to afford optimal separation of phosphorylated and nonphosphorylated peptides. All assays were performed in 50 mM Hepes (pH 7.4), 0.015% (v/v) Brij-35, and 5 mM MgCl2, and the real-time or end point degree of peptide phosphorylation was calculated by differentiating the ratio of the phosphopeptide:peptide present in the reaction. Kinase activity in the presence of different redox reagents was quantified by monitoring the generation of phosphopeptide during the assay, relative to controls. Data were normalized with respect to control assays, with phosphate incorporation into the peptide generally limited to <20% to prevent depletion of ATP and to ensure assay linearity. ATP KM and the concentration of a compound that caused IC50 values were determined by nonlinear regression analysis using GraphPad Prism software. Where specified, kinase assays using Aurora A were supplemented with 100 nM GST-TPX2 or GST alone. Assays for CAMK kinases included 5 mM CaCl2 and 2 μM calmodulin as standard. Where appropriate, PKG1 assays were performed in the presence of 1 mM cGMP. Recovery of Aurora A activity from oxidative inhibition was assessed by monitoring substrate phosphorylation in the presence of peroxide in real time, followed by subsequent introduction of DTT or GSH. To standardize this real-time reversible redox regulation assay for all kinases, enzymes were preincubated in the presence or absence of 5 mM H2O2 on ice for 30 min, and then substrate phosphorylation was initiated with the addition of 1 mM ATP and the appropriate substrate peptide in the presence (where indicated) of 10 mM DTT unless otherwise stated. Aurora A kinase assays were also developed with recombinant GST-TACC3 as a substrate. Kinase assays where initiated after the addition of GST-TACC3 (1 μg) in the presence of 0.5 mM ATP and 5 mM MgCl2 and TACC3 Ser558 phosphorylation were detected by immunoblotting with a pSer558 TACC3 antibody after 15-min assay time, as previously described (74). Reactions were terminated by the addition of SDS loading buffer. Equal loading of TACC3 substrate and Aurora A was confirmed with an anti-TACC3 antibody and antibodies for total and phosphorylated Aurora A (pThr288). Aurora A autophosphorylation after phosphatase treatment was also detected using a previously described Thr288 phospho-specific antibody (74). Dephosphorylated Aurora A (1 μg) was produced by coexpression with λPP in E. coli and then incubated with 1 mM ATP and 10 mM MgCl2 for the indicated time periods under reducing (+1 mM DTT) or oxidizing (+1 mM H2O2) conditions in the presence and absence of 1 μg of GST-TPX2, 1 μg of GST, or 100 μM MLN8237. Reactions were terminated by the addition of SDS gel-loading buffer.

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