Figure 4.

ORF8 mediates the downregulation of the spike protein through multiple pathways. (A,B) HEK293T cells were cotransfected with the spike-encoding plasmid together with the pLAS2-ORF8-IRES-GFP (ORF8) or pLAS2-IRES-GFP (EV) plasmid at a ratio of 1–3. Forty-eight hours after transfection, the cells were treated with DMSO, MG132 (20 μM), DBeQ (15 μM), bafilomycin A1 (Baf A1; 200 nM), chloroquine (20 μM), and NH₄Cl (20 mM) for 24 h, and the cells were collected for flow cytometry using antibodies against the spike protein and HLA-A2. By using this method, we were allowed to analyze the cells with successful transfection by gating the GFP-positive cell population. Here, the results of the spike protein are shown as (A) histograms comparing EV- or ORF8-transfected cells of one experiment and (B) the scatter plot figure of the results from six-independent transfection. (C) Immunoblots of cell lysates prepared from one experiment using antibodies against the spike protein, FLAG tag, and actin. The bar figure in (B) shows the means ± SDs (error bars) and unpaired Student’s t-test was used, and p < 0.05 indicates a statistically significant difference; *p < 0.05, **p < 0.01, ***p < 0.001.