Journal: The Journal of Biological Chemistry
Article Title: Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors
Figure Lengend Snippet: P472L mutation does not disrupt the binding sites of p97 inhibitors. A, ITC was used to measure the binding of CB-5083 to p97 and the P472L mutant. Top panels are raw data, and lower panels show isotherms with fitted curves and representative K D values. B, effect of increasing NMS-873 concentrations on TR-FRET generated by p97 and the P472L mutant in the presence of BODIPY-FL–ATP, and a terbium-labeled anti-His tag antibody was measured ( n = 4, S.D.). Apparent K D values extrapolated from NMS-873–dependent increases in TR-FRET are shown. C, fluorescence polarization experiments were performed ( n = 2, S.D.) to measure the enhanced binding of EDA–ADP–ATTO-495 to p97 and the P472L mutant with increasing NMS-873 concentrations. Apparent K D values with S.E. are shown.
Article Snippet: The 2-fold serial dilutions of p97 proteins (maximum 40 μ m , purified from bacteria) were mixed with 20 n m BODIPY-FL–ATP (ThermoFisher Scientific) or 10 n m EDA–ADP–ATTO-495 (Jena Bioscience) in black low volume 384-well plates.
Techniques: Mutagenesis, Binding Assay, Generated, Labeling, Fluorescence