Figure 3.

Site-directed mutagenesis identifies residues essential for TC formation. The effect of specific point mutations within the Pip ID was analyzed by gel retardation assay on the λ1B (A), kE3′ (C), and ISG15 (D) probes. Proteins were prepared by in vitro transcription/translation and equal amounts of PU.1 and wt Pip or mutants were incubated with DNA probes as indicated. Binding of PU.1 as a monomer in A is indicated by an arrow. (B) The amount of TC formed by the different Pip mutants was quantitated by using a PhosphorImager and is represented as a percentage relative to wt Pip. The results of a representative experiment are shown.