FIGURE 5.

Analysis of CHERP truncation mutants defines a region responsible for nuclear targeting. A, schematic overview of CHERP truncation mutants (symbols as defined in Fig. 3A). The GFP moiety was not depicted graphically. B, Western blot (WB) of cytoplasmic and nuclear fractions prepared from HEK293 cells transfected with the indicated CHERP truncation mutants or GFP alone and probed with a GFP antibody (left) or a CREB antibody (right). C, confocal live cell images show nuclear localization of wild-type CHERP (CHERP(1–916)-GFP), a NH₂-terminal truncation mutant (CHERP(342–916)-GFP), and a COOH-terminal truncation (CHERP(1–725)-GFP). D, representative confocal images of a HEK293 cell co-transfected with CHERP(1–725)-GFP and DsRed2-ER that was subsequently stained with DAPI (blue). E, top, representative Western blot (GFP antibody) using WCL samples from HEK293 cells (mock transfected versus transfection of indicated constructs) confirmed expression of exogenous CHERP proteins. Bottom, schematic of a series of NH₂- and COOH-terminal truncation mutants of CHERP. F, live cell confocal images of GFP-tagged CHERP truncation constructs expressed in HEK293 cells. The parental GFP vector was used as a control (GFP). G, quantitative analysis of CHERP construct distribution from confocal images, expressed as a ratio (N:C) on a logarithmic scale of fluorescence intensity in comparable nuclear and cytoplasmic regions of interest (*, p < 0.01).