Skip to main content
. 2023 Jun 23;299(8):104955. doi: 10.1016/j.jbc.2023.104955

Figure 1.

Figure 1

SARS-CoV-2 ORF8 is an ER luminal protein.A, amino acid sequence analysis (Protter) of ORF8 and prediction as an ER luminal protein (due to the presence of ER signal peptide and the absence of a transmembrane domain). B, A549 cells transfected with a plasmid encoding ORF8-Strep were fixed, permeabilized, and immunostained for Strep (ORF8) or PDI (ER marker), which were analyzed by fluorescence confocal microscopy imaging. The cells were counterstained using DAPI and phalloidin (upper left). The dashed box is digitally enlarged to show colocalization (bottom right) of ORF8 (upper right) and ER (bottom left). White scale bars represent 10 μm. C, the pixel intensities of ORF8 and PDI along the dashed arrow in (B) are plotted. D, HEK293T cells transfected with a plasmid-encoding ORF8-Flag were mechanically lysed and fractionated by differential centrifugation. The indicated subcellular fractions were evaluated by immunoblot analysis for Flag (ORF8), Calnexin (ER marker), COX4 (mitochondrial marker), or β-actin (cytosolic marker). E, the ER subcellular fractions in (D) were further incubated with two concentrations of digitonin (0, 0.035, or 0.2%). The fractions were then centrifuged, and the supernatants containing digitonin-solubilized proteins were evaluated by immunoblot analysis as in (D). B–E, the data represent three independent experiments. ER, endoplasmic reticulum; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.