Hl344 gtx635679

HEK293T cells were seeded in a 12-well plate at a density of 2 × 10⁵ cells/well and cultured for 24 h. Next, the cells were transiently transfected with each spike plasmid and cultured for two days. Thereafter, the cells were lysed using ULTRARIPA A buffer (BioDynamics Laboratory Inc., Tokyo, Japan). Cell lysates were treated with PAA at the indicated concentration for 10 min. Then, the amount of protein in the cell lysates was measured using BCA protein assay kit (Takara Bio Inc., Shiga, Japan). Equal amount of protein was determined by SDS-PAGE and electrotransferred to an Immobilon-P PVDF membrane (EMD Millipore, Billerica, MA, USA). Western blotting was performed using the standard method, and the following antibodies were used: anti-SARS-CoV-2 spike (1A9, GTX632604, mouse monoclonal antibody, GeneTex, Irvine, CA, USA), anti-SARS-CoV-2 nucleocapsid (HL344, GTX635679, rabbit monoclonal antibody, GeneTex), anti-GAPDH (3H12, M171-3, mouse monoclonal antibody, MBL), and horseradish peroxidase-conjugated sheep anti-mouse IgG (NA931, Amersham Biosciences, Amersham, UK). Immunoblot signals were developed using EzWestLumi plus (ATTO Corp., Tokyo, Japan) and recorded with an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Tokyo, Japan).