Ei24 can inhibit nuclear translocation of p53. (A) Schematic of the experimental layout. (B) Western analysis of lysates from p53 WT and KO MEFs, treated with either 50 µM etoposide or DMSO vehicle control for 16 h, using an anti-Ei24 antibody with α/β-tubulin as a loading control. (C) Cells as in A were imaged live by CLSM 8 h after transfection. Cytoplasmic aggregates of GFP-p53 are indicated by yellow arrows. Bar, 20 µm. (D) Digitized images such as those in C were analyzed to calculate the nuclear-to-cytoplasmic fluorescence ratio (Fn/c; see Materials and methods). Results are for the mean ± SEM (error bars; n ≥ 34) from a single assay representative of three separate experiments. (E) HeLa-Bcl_XL cells transfected to express DsRed2-fusion proteins, as indicated, were fixed 20 h after transfection before immunostaining using specific antibodies for endogenous p53 (top) or hnRNPA1 (bottom), and DAPI counterstaining. Bars, 20 µm. (F) Results from analysis such as that shown in E are for the mean ± SEM (error bars; n ≥ 65). P-values denote significant differences.