FIGURE 2.

Translation-independent ER localization is mediated by the ALPP ORF. COS-7 cells were transfected with plasmids containing either ALPP, t-ftz, or the AF1, AF2 fusion constructs and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (Ctrl), puromycin (Puro), or HHT for 30 min and then extracted with either digitonin alone, or for puromycin-treated cells, with 20 mm EDTA. The cells were then fixed, stained for mRNA using specific FISH probes (ALPP probe for AF1, ftz probe for AF2), and imaged. A, representative FISH images of cells expressing AF1 or AF2. B, quantification of the fluorescence intensities of mRNA in the ER and nucleus. All data were normalized to the ER staining intensity in the control treated group for each construct. Each bar represents the average ± S.E. (error bars) of three independent experiments (each experiment consisting of n>30 cells). Scale bar, 20 μm.