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. 2022 Jun 28;54:102388. doi: 10.1016/j.redox.2022.102388

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 1 — ORF8 is an ER luminal protein.

(A) Schematic presentation of full-length SARS-CoV-2 ORF8.

(B) Immunoblotting of ORF8 in cell extracts and FLAG immunoprecipitates from the culture medium (CM) of HEK 293T, HepG2, HeLa and Vero E6 cells overexpressing ORF8-FLAG.

(C) Immunofluorescent analysis of ORF8 localization. HEK 293T cells expressing ORF8-FLAG were immunostained using antibodies against FLAG, PDI (ER marker) and GM130 (Golgi marker). Scale bars, 10 μm.

(D) ORF8 presented in the soluble fractions. The total lysates (TL) were separated into cytosol (Cyto) and total membrane (TM) fractions. The TM fractions were extracted with Na₂CO₃ and separated into soluble (S) and pelleted membrane (P) fractions. FLAG, BiP, calreticulin, calnexin and α-tubulin were immunoblotted.

(E) Fluorescent photomicrographs of HepG2 cells expressing ORF8-FLAG, which were permeabilized with digitonin, and then digested with proteinase K in the absence or presence of Triton X-100. Endogenous PDI was immunostained (red) as an ER luminal protein marker. Scale bars, 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)