Transfections were performed according to the TransIT-mRNA protocol (Mirus Bio MIR 2225). Cells were plated at 300,000 cells/mL in a 24-well tissue culture plate for western blot detection and 80,000 cells/mL in an eight-well Nunc™ Lab-Tek™ II Chamber Slide™ System (Thermo Fisher 154534) for immunocytochemistry. Cells were incubated at 37 °C with 5% carbon dioxide for 24 h. Following this incubation, the cells were transfected with 0.5 µg/well mRNA or 1.0 µg/well mRNA-LNP for western blot detection or 0.18 µg/well mRNA for immunocytochemistry, then returned to 37 °C with 5% carbon dioxide. Transfected cells were harvested at 8, 24, and 48 h for western blot detection or at 16 h for immunocytochemistry. A negative control with TransIT reagents alone (no mRNA) was included to provide background detection levels.

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