Fig. 5. Zfp488 exhibits nuclear localization and has transcriptional repression activity. Cells transfected with expression vectors (pCS2) for Myc-tagged Zfp488 and its various mutants were assessed for nuclear localization defined by DAPI staining and for Zfp488 transcription activity in an in vitro assay. (A,B) Nuclear localization of Myc-tag staining (arrows) is detected in both COS7 and NIH3T3 cells when transfected with Myc-Zfp488 and analyzed by indirect immunofluorescence using anti-Myc antibody (red) together with DAPI (blue) to delineate the nucleus. (C-F) COS7 cells transfected with expression vectors for Myc-tagged proteins of Zfp488 full-length (C), Zfp488F2 (D), Zfp488F12 (E) and ZF12 (F) domain only were examined for Myc (red) and DAPI (blue) immunofluorescence. Arrows indicate expression of Myc-Zfp488 and its derivatives. (G) A schematic diagram shows the in vitro assay for Zfp488 transcription activity. (H) NIH3T3 cells were transiently transfected with L8G5-luc reporter (Lu et al., 1999) and expression vectors encoding LexA-VP16, GAL4-Zfp488 or its truncated forms, as indicated. Transfection of GAL4-Zfp488 resulted in repression of luciferase reporter activity induced by LexA-VP16 in this assay. The Zfp488 construct lacking both zinc-finger motifs (Zfp488ZF12) and its N-terminal fragments 1-184 (Zfp488N184) and 1-69 (Zfp488N69), as well as its zinc-finger domain only segment (Zfp488ZF12), were fused in-frame with GAL4 in an expression vector. The relative activity of GAL4-Zfp488 truncated derivatives was normalized to that of LexA-VP16 activation. At least three independent transfection experiments were performed and data are presented as the mean±s.d.