Figure 5.

Like VP16, ZF binds to HCF but not to the mutant HCF(P134S), as determined by GST pull-down assay (A and B) and by mammalian two-hybrid assay (C). GST and GST fusion proteins of ZF and VP16 were produced in E.coli strain BL21(DE3), coupled to Sepharose beads. HCF was labeled with [³⁵S]methionine by in vitro transcription and translation in a rabbit reticulocyte system (TnT; Promega). The protein-bound beads were incubated with an equivalent amount of ³⁵S-labeled HCF (A) or HCF(P134S) (B). After extensive washing, proteins were eluted, fractionated by 10% SDS–PAGE and visualized by autoradiography. The first lane on the figure represents one-tenth of the input. Plasmids expressing GAL–HCF, GAL–HCF(P134) and GAL DBD alone (C) were co-transfected into COS7 cells with either pcZF, pcLuman or their parental vector pcDNA3 (0.5 µg each), with pG5EC as the reporter. The CAT activity was measured 48 h post-transfection.