FIGURE 2.

Mapping the putative interaction regions on hZimp7 and PIAS3 proteins. A, full-length or truncated fragments of Zimp7 were fused to Gal4 DBD in the pM vector. Numbers correspond to amino acid residues. CV-1 cells were cotransfected with pM vectors, luciferase reporter constructs, with or without PIAS3 plasmids. B, HEK293 cells were transfected with pcDNA3-FLAG-tagged full-length or truncated Zimp7 (1 μg) with HA-tagged PIAS3 (1 μg). Equal amounts of the whole cell lysates were used for immunoprecipitation (IP) with an anti-FLAG monoclonal antibody. Immunoprecipitates were then analyzed by immunoblotting (IB) and probed by Zimp7 and PIAS3 antibodies. C, a schematic representation of the domains of human PIAS3 protein. Numbers correspond to amino acid residues. D, equal amounts of GST fusion proteins were used to pull down endogenous Zimp7 protein in HEK293 cells. GST protein alone was used as a negative control. Equal amounts of the above GST proteins were analyzed on SDS-PAGE. Material bound to the GST columns was subjected to SDS-PAGE and analyzed by Western blot with a Zimp7-specific antibody.