4 to 15 sds polyacrylamide gels

293T cells [American Type Culture Collection (ATCC), CRL-3216] in six-well plates were directly transfected with 2 μg of mRNA-N-LNP or not transfected (as a cell-only control). Eighteen hours after transfection, cells were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) for Western blot analysis. Cell lysates were centrifuged, followed by collection of supernatants for quantification of total protein concentration using Microplate bicinchoninic acid protein assay kit (Pierce, Thermo Fisher Scientific). Equal amounts of protein were separated by SDS–polyacrylamide gel electrophoresis using 4 to 15% SDS polyacrylamide gels (Bio-Rad). Proteins were transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked in tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST; Thermo Fisher Scientific) and 5% (w/v) nonfat dried milk (Bio-Rad) for 1 hour at room temperature, followed by incubation with anti–SARS-CoV-2 nucleocapsid mouse monoclonal antibody (MA5–29981, Thermo Fisher Scientific; 1:1000) overnight at 4°C. After washing in TBST (three times for 5 min), the membrane was incubated for 1 hour with horseradish peroxidase (HRP)–linked anti-mouse IgG (1:5000; 7076S, Cell Signaling). The membrane was washed, and proteins were visualized using the enhanced chemiluminescence Western blotting substrate (Thermo Fisher Scientific).