Figure 5. Contributions of AFF4 ELLBow interactions to binding in solution.

(a) Binding of AFF4^ELLBow wild type (WT) and mutants in the N-terminal α-helix to Sumo-ELL2^Occ. Sumo-ELL2^Occ binding to fluorescently labelled AFF1^ELLBow peptide 358–390 is competitively inhibited by increasing amounts of AFF4^ELLBow, as described in experimental procedures. Error bars reflect the s.e. from three experimental replicates in a,b. (b) Binding of AFF4^ELLBow WT and mutants in the central cluster and elbow joint, assayed as in a. (c) GST fusions of the indicated ELL2^Occ mutants were immobilized and their ability to pull-down His₆-tagged WT AFF4^ELLBow assessed. An uncropped version of this pull-down gel is shown in Supplementary Fig. 4. (d) Direct binding of WT and mutant Sumo-ELL2 to fluorescently labelled AFF1^ELLBow peptide. The assay was performed in triplicate. The s.e. from the three replicates in d is smaller than the symbols used to plot the data points.