Plk1 pbd

FLIPs and peptides to be tested were dissolved in DMSO (10 mM) and diluted to working concentrations in assay buffer (a maximum of 600 μM, which equates to a maximum 6% DMSO tolerance determined for the assay). Assays were optimized following standard guidelines (http://www.ncbi.nlm.nih.gov/books/NBK92000/). The PLK1 PBD (367–603) and PLK3 PBD (335–646) proteins were obtained from BPS Bioscience Inc. (San Diego, CA); 41.4 nM PLK1 and 245 nM PLK3 were used per reaction. The fluorescein-tracer phospho-peptides (MAGPMQS[pT]PLNGAKK for PLK1, and GPLATS[pT]PKNG for PLK3) were used at a final concentration of 10 nM. Incubation was carried out at room temperature for 45 min on a shaker. Fluorescence was measured using either a DTX 880 plate reader and Multimode Analysis software (Beckman Coulter, now Molecular Devices, Brea, CA) or a SpectraMax i3 (Molecular Devices, Brea, CA). The polarization values in millipolarization (mP) units were measured at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. Each data point was performed in triplicate for every experiment, and experiments were performed at least three times. An IC₅₀ value for each compound was calculated from non-linear regression analysis of the plots of mP values relative to PBD-tracer mP values alone versus FLIP/peptide concentrations (Supplementary Figures 8, 9).

ABBAs were dissolved in DMSO (10 mM) and diluted to working in assay buffer to a maximum concentration of 600 mM (maximum of 6% DMSO tolerance in the assay). The PLK1 PBD (367–603) and PLK3 PBD (335–646) proteins were obtained from BPS Bioscience Inc. (San Diego, CA); 17 ng PLK1 and 156 ng PLK3 were used per reaction. The fluorescein-tracer phospho-peptides (MAGPMQS[pT] PLNGAKK for PLK1, and GPLATS[pT]PKNG for PLK3) were used at a final concentration of 10 nM. Incubation was carried out at room temperature for 45 min on a shaker. Fluorescence was measured using a DTX 880 plate reader and Multimode Analysis software (Beckman Coulter, Brea, CA). The polarization values in millipolarization (mP) units were measured at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. Each data point was performed in triplicate for every experiment, and experiments were performed at least three times. IC₅₀ values were calculated from non-linear regression analysis (GraphPad Prism) of the plots of mP values relative to PBD-tracer mP values alone versus PBD-inhibitor concentrations.