PCET mutations impair KIX binding. (A) Representative binding curves showing the change in fluorescence anisotropy of 5-carboxyfluorescein tagged HEB-PCET (12-25) peptide with increasing total concentration of either wild-type (●) or Lys667Glu mutant (○) KIX. (B) In a mammalian 2-hybrid assay, the KIX domain (residues 586-673 of CBP) fused to a GAL4 DNA-binding domain can recruit E2A to the GAL4 binding sites on a reporter plasmid, driving expression of firefly luciferase. The luciferase expression was normalized against expression of Renilla driven by a constitutively active internal control plasmid used to control for variations in transfection efficiency. Renilla-normalized luciferase expression of E2A (1-483) PCET mutants were measured relative to that seen with wild-type E2A (1-483), with the result presented being the mean from at least 3 independent transfections ± the SD (represented by vertical error bars). #P < .015 vs wild-type 2 × VP16-E2A (1-483). *P < .001 vs wild-type 2 × VP16-E2A (1-483). The results shown in the figure are the mean value from at least 3 independent transfections; error bars represent SD. Statistical significance was measured using a 1-way ANOVA with Tukey post hoc test. (C) SV293T cells were transfected with FLAG-CBP and GAL4-E2A (1-483) or GAL4-E2A (1-483) PCET mutants. Lysates were subjected to immunoprecipitation with anti-FLAG and immunoblotted with anti-E2A yae or anti-FLAG.