To generate SARS‐CoV‐2‐infected/mock‐infected cell pellets for positive/negative control staining, African green monkey (Vero E6) cells (ATCC, American Type Culture Collection, Rockville, MD, USA) were incubated with SARS‐CoV‐2 D614G virus (hCoV‐19/England/IC19/2020 (EPI_ISL_475572)) diluted in Dulbecco's Modified Eagle's Medium (DMEM) at a multiplicity of infection (MOI) of 3 (or with plain media for mock‐infected cells) for 1 h at 37 °C, after which DMEM was removed and replaced with fresh medium supplemented with 10% foetal bovine serum (Labtech, Heathfield, UK, 1% nonessential amino acids and 1% penicillin–streptomycin; Gibco, Thermo Fisher Scientific). Cells were collected 48 h after infection or mock‐infection by scraping, washed in ice‐cold PBS twice, and fixed in formalin at room temperature for 24 h. Fixed cells were washed twice in PBS, centrifuged, and the pellet was processed using the Thermo Scientific Shandon Cytoblock system according to the manufacturer's instructions.
For protein lysate preparation, infections were performed as above but at an MOI of 0.1. Cells were collected by scraping 48 h postinfection, pelleted, and lysed in 500 μl ice‐cold RIPA buffer on ice for 30 min. Lysates were clarified by centrifugation at 4 °C for 30 min, then heated at 90 °C for 10 min in Laemmli buffer (BioRad, Hercules, CA, USA) with 10% β‐mercaptoethanol (Sigma‐Aldrich).
For protein lysate preparation, infections were performed as above but at an MOI of 0.1. Cells were collected by scraping 48 h postinfection, pelleted, and lysed in 500 μl ice‐cold RIPA buffer on ice for 30 min. Lysates were clarified by centrifugation at 4 °C for 30 min, then heated at 90 °C for 10 min in Laemmli buffer (BioRad, Hercules, CA, USA) with 10% β‐mercaptoethanol (Sigma‐Aldrich).