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.

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