FIGURE 6.

SUMOylation kinetics and [ATP] dependence. These data were used to establish the standard assay conditions and for subsequent experiments relating to NBD1 conformation. A, time courses of WT and F508del NBD1 modification by SUMO-1 at 0.1 or 2 mm ATP at 27 °C. Immunoblots were performed using NBD1 (antibody 660) antibody. Quantitation of the data is provided to the right, where density values were normalized to values at 5 min; the mean data are derived from three independent experiments. B, in vitro SUMOylation of WT or F508del NBD1 at three levels of purified protein, run for 1 h at 27 °C. The indicated reactions included preheating to 30 °C for 30 min prior to adding the SUMOylation reagents for the standard assay. Controls were performed without added E1, E2, or ATP (left and right lanes).