Isothermal Titration Calorimetry (ITC).
ITC measurements were carried out at 25℃ on a Nano ITC calorimeter . Proteins were extensively dialyzed against 20 mM sodium phosphate, pH 7.0, 200 mM KCl, 5mM β-mercaptoethanol (β-ME), and degassed for 15 min before each experiment. Full-length VCP was placed in the syringe and titrated as ligands into the sample cell containing VCP. The first technical injection of 0.4 l was followed by 25 injections of 2 l into the sample cell (300 l), using 250 rpm stirring and a 200-s delay between successive injections. At least three titration experiments were performed for each ligand, and the data were corrected for heats of dilution by injecting the same ligand concentration into a matched buffer. Corrected data were analyzed with a one-site binding model and nonlinear least-squares fitting to derive the KD value, using the NanoAnalyze 3.5.0 software package.