Fig. 6: Bora and Tpx21–43 compete for AURKA binding.
a Schematics of a competitive displacement binding assay using fluorescein-labeled Tpx21–43 (FITC-Tpx21–43) as a probe. Complex formation between FITC-labeled Tpx21–43 polypeptide and AURKA followed by the disassembly of the FITC-Tpx21–43/AURKA complex by increasing amount of competitor (cold Tpx21–43, Bora, or pBora) was monitored by fluorescence polarization. b Binding of fluorescein-labeled Tpx21–43 polypeptide to AURKAWT assessed by monitoring the fluorescence polarization signal in the presence of increasing concentrations of AURKAWT. Kd value represents mean value with standard deviations of the mean as error bars (n = 3 independent experiment samples). c Competitive binding assay where fluorescein-labeled Tpx21–43 polypeptide in complex with AURKAWT is displaced by increasing amount of competitor (cold Tpx21–43, Bora1–224, or pBora1–224) and monitored by fluorescence polarization signal. Displayed data points and the half maximal inhibitory concentration (IC50) value represent the average fluorescence polarization for each reaction conditions with standard deviations of the mean as error bars (n = 3 independent experiment samples). ND: not determined. d Binding of fluorescein-labeled Tpx21–43 polypeptide to AURKAT288V assessed by monitoring the fluorescence polarization signal in the presence of increasing concentrations of AURKAT288V. Kd value represents mean value with standard deviations of the mean as error bars (n = 3 independent experiment samples). e, f Competitive binding assay where fluorescein-labeled Tpx21–43 polypeptide in complex with AURKAT288V is displaced by increasing amount of cold Tpx2, Bora1–224, or pBora1–224 competitor (e), or by increasing amounts of cold pBora1–224, pBora18–120, or the indicated pBora18–120 mutants in the M1 and M2 motifs. Data presented as in c. g Activation of AURKAWT ATPase activity by Bora1–224, pBora1–224, and Tpx21–43 as assessed using the ADP-Glo assay with Kemptide substrate. Displayed data points and EC50 values represent the average luminescence for each reaction condition with standard deviations of the mean as error bars (n = 3 independent experiment samples). RLU: relative light unit. h Activation of AURKAT288V ATPase activity by Bora1–224, pBora1–224, and Tpx21–43 as assessed using the ADP-Glo assay with Kemptide substrate. Data presented as in g. i Activity of the AURKAT288V/pBora1–224 complex in the presence of increasing concentration of competitor Tpx21–43 or Bora1–224 assessed using the ADP-Glo assay in the presence of Kemptide substrate. Displayed data points and the half maximal inhibitory concentration (IC50) value represent the average fluorescence luminescence for each reaction conditions with standard deviations of the mean as error bars (n = 3 independent experiment samples). ND: not determined.
