Figure 1
AE but not AE9aNT forms a heterodimer with CBFβ but both proteins bind to DNA. (a) Representation of the proteins used in this study: Full-length AE, AEtr, splice-variant AE9a and DNA-binding Runt domain of AML1. The arrows indicate the location of the two mutations within the Runt domain, asparagine 109 (N109) and threonine 161 (T161) to alanines. (b) Coimmunoprecipitation experiments of endogenous CBFβ together with wt, single mutant (N or T) or double mutant (NT) of Runt, AE, AEtr or AE9a. The wt proteins (runt, AE, AEtr and AE9a) strongly interact with endogenous CBFβ (upper panels, lanes 1, 5, 9 and 13), whereas single N or T mutants interact only weakly (upper panels, lanes 2, 3, 6, 7, 10 and 11); the NT double mutant no longer binds to CBFβ (upper panels, lanes 4, 8, 12 and 14). Input controls are shown in the lower panels. Flag-ETO was used as negative control (lane 15). The asterisk denotes the light chain of the antibody used for immunoprecipitation. (c) Electrophoretic mobility shift assays showing that Runt and Runt NT (lanes 3 to 6) as well as AE9a and AE9aNT (lanes 11 to 14) bind to DNA (see label ‘A’). Addition of an anti-Flag antibody resulted in super shifted complexes (see label ‘B’). Flag-tagged protein expression was verified by western blot shown in Supplementary Figures S1d and e.
