Figure 1

Generation of murine and human -HA tagged RAI1 and molecular evaluation of the resulting proteins. A) Schematic representation of mouse (Rai1) and human (RAI1) genomic and protein structure. In blue is represented the Poly-Gln domain, in yellow: Poly-Ser domains, in black: in silico described NLS, in slanted lines: the PHD domain. The coding sequence for HA epitope (represented in red) was added by PCR at the 3' end of full-length cDNA. B) Cells transfected with either mouse Rai1-HA plasmid (Rai1-HA), the human RAI1 plasmid, RAI1 or RAI1-HA, an empty vector (e/v) or untransfected cells (u/t) were lysed and a western blot analysis was performed with an anti HA antibody (αHA). The molecular weight of the resulting proteins is depicted. The anti β-tubulin antibody (α β-tubulin) was used as loading control. C) Neuro-2a cells were transiently transfected with the mouse Rai1-HA plasmid (Rai1-HA), the human RAI1 and RAI1-HA plasmids. Immunofluorescence with an antibody that recognized the HA tag (αHA) (red) and an antibody that recognizes the first 30 aa of RAI1 (αRAI1) (green) are shown. Untransfected cells in the same slide were used as negative control. Nuclei were stained with DAPI. The table represents the subcellular localization observed for 200 counted cells positive for immunodetection. D) Transactivational activity. The fold of luciferase activation is represented for the empty vector (gray), Neuro-2a cells transfected with mouse Rai1-HA (black), human RAI1 (light blue), and RAI1-HA (light green) and HeLa cells transfected with Rai1-HA (white). Values represent mean +/- SEM.