Figure 4: TAF8–TAF2 interactions.

(a) His-tagged TAF2 binding to overlapping peptides of the TAF8 C-terminal region (residues 105–310) spotted onto nitrocellulose membranes (spots A2-G4, left) was analysed by utilizing a peptide array. Bound TAF2 was visualized by luminol reaction and signal intensities were plotted for each spot after background subtraction (right). Spots A1, G5 and G6 served as positive controls. TAF2 protein was omitted for the control membrane. The four major binding regions (I–IV) are indicated above the histogram. (b) SPR experiments with immobilized full-length TAF2 as ligand and MBP (control) as well as MBP fusions of TAF8 fragments 105–310, 141–310, 200–310 and 105–260 as analytes. TAF8 deletion constructs are schematically shown as bar diagrams (left). TAF2-interacting regions on TAF8 as identified in a are highlighted. SPR sensorgrams at identical analyte concentrations of 500 nM are plotted as RU versus time (right). (c) SEC analyses assessing the influence of TAF8 point mutations on TAF2 binding. Elution profiles for the indicated proteins and protein complexes are plotted on the left and SDS–PAGE analyses of each run are shown on the right. Molecular masses of protein standards are denoted on the left of the gels and protein names on the right.