2 mercaptoethanol

Western blotting targeting the S1 subunit of the S protein, the S2 subunit of the S protein, or the N protein was performed as previously described [32 (link)]. Briefly, SARS-CoV-2 solution (~7.0 log₁₀ TCID₅₀/mL), or each recombinant viral protein, was mixed with an equal volume of dextrin or TY-1. The final concentrations of dextrin and TY-1 in the mixture were 2.5 and 5.0 mg/mL, respectively. The final concentration of recombinant proteins in the mixture was 10 μg/mL. These mixtures were combined with sodium dodecyl sulfate (SDS) buffer with 2-mercaptoethanol (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan) either immediately (0-h reaction time) or were incubated at 25 °C for 24 h prior to adding the SDS buffer (24-h reaction time). These samples were subjected to SDS–polyacrylamide gel electrophoresis using Mini-PROTEAN^® 3 Cell (Bio-Rad Laboratories Inc., Hercules, CA) and Power PAC^TM HC power supply (Bio-Rad Laboratories Inc.), and western blotting using Power PAC^TM HC power supply and Luminescent Image Analyzer LAS-3000 (FUJIFILM Co. Ltd., Tokyo, Japan) to detect the S1 subunit of the S protein, the S2 subunit of the S protein, and the N protein.

The miPS cells used in the present study (APS0001; iPS-MEF-Ng-20D-17 mouse-induced pluripotent stem cell line) were purchased from RIKEN BRC (Ibaraki, Japan) [22 (link)]. The cells were incubated with inactivated murine embryonic fibroblast (MEF) feeder cells in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 15% KnockOut™ Serum Replacement (Invitrogen), 1% nonessential amino acids (Chemicon, Temecula, CA, USA), 1% l-glutamine (Chemicon), 1000 U/mL penicillin–streptomycin (P/S; Invitrogen), and 0.11 mM 2-mercaptoethanol (Wako Pure Chemical Industries Ltd., Osaka, Japan); 60-mm cell culture plates were used for passaging the cells at a density of 1 × 10⁵ cells/plate. Cells were grown in 5% CO₂ at 95% humidity, and the culture medium was changed each day.