Figure 5.
Binding affinity of Cm eIF4E and Cm eIF4G1003-1092. MBP pull-down assays showing the interaction of purified Cm eIF4E (wild type, full length; increasing concentrations, 0.5–3 μm) and MBP-tagged Cm eIF4G1003-1092 (wild type or mutant in bacterial lysates at a fixed concentration). A, Pull-down with Cm eIF4G1003-1092-WT. B, Pull-down with MBP used as a negative control. C to E, Pull-down with MBP-eIF4G1003-1092 mutants in C, NC, and C-NC domains. EIF4G lysates (wild type and mutants) were incubated with purified Cm eIF4E at concentrations of 0.5, 1, 2, and 3 μm (corresponding to lanes 1, 2, 3, and 4 of each gel, respectively) in a volume of 300 μL. The input samples (lanes 1 to 4) and pull-down fractions (lanes 5 to 8) were analyzed in a 12% SDS-PAGE followed by Coomassie Brilliant Blue staining, loading in gels 5% and 16% of the total volumes, respectively. F, Graph representing the specific binding of Cm eIF4E with Cm eIF4G1003-1092 (wild type and mutants), plotted as a function of Cm eIF4E concentration. Data points in the binding curves represent means calculated from data points of two different experiments as in A, C, and D. Each data was fitted using a one-site binding model.