Fig. 5. Kinetics analyses of the association of GST-WW2 of Nedd4 with the phosphorylated and non-phosphorylated extended PY motif of Cx43. (A) Plot of steady-state binding of Cx43 CT_phosph () and Cx43 CT () against GST-WW2 concentration. The lines represent the best fit to the equation RU=RU_max/(1+K_Dapp/S). RU and RU_max are the resonance and the maximal resonance of bound GST-WW2, S is its free concentration, and K_Dapp is the apparent equilibrium dissociation constant. K_Dapp values (in µM) are listed as means ± s.e.m. in the table. NB, no binding. (B) GST-WW2 fusion protein (0.39 µM) was pre-incubated with increasing concentrations of non-biotinylated Cx43 CT_phosph peptide, and the mixture was applied to a sensor chip with immobilized Cx43 CT_phosph peptide at its surface. The steady-state signal B was expressed as B/B_max, where B_max is the steady-state signal at zero competing peptide. Binding of GST-WW2 to the immobilized Cx43 CT_phosph decreased with increasing amounts of competing peptides.