FIG. 9.

Expression of constitutively active MKK1 enhances the activity of topoisomerase IIα immunoprecipitated from nuclear extracts. NIH 3T3 cells transiently transfected with wild-type MKK1 or MKK1-G1C were used to prepare nuclear extracts. Topoisomerase IIα was immunoprecipitated from these extracts, and its activity was determined by measuring kinetoplast decatenation over time. (A) Immunoblots of topoisomerase IIα immunoprecipitated from nuclear extracts transfected with wild-type (WT) or constitutively active MKK1. (B) Immunoblots of ERK2 in nuclear extracts, showing gel mobility retardation of ERK2 (ppERK) in cells transfected with MKK1-G1C. (C) Decatenation of DNA versus time, measured after immunoprecipitation of topoisomerase IIα from nuclear extracts of cells transfected with MKK1-G1C (closed circles) or wild-type MKK1 (open circles). (D) Decatenation assay used to quantify the amount of decatenated kinetoplast DNA in panel C. Shown are catenated DNA substrate (C) and decatenated product (D) on an ethidium bromide-stained agarose gel. Data are representative of two independent transfections.