ORF1p Quantification by Western Blot

Cells were lysed in RIPA buffer with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na₃VO₄ by sonication. Extracts were centrifuged at 14,000 r.p.m (18,407 × g) for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 4–12% Criterion^TM XT Bis-Tris Protein Gel (Biorad), transferred by Trans-Blot-Turbo (Biorad), and blocked with EveryBlot Blocking Buffer (Biorad). Primary antibodies were incubated with membranes overnight at 4 °C. Antibody against ORF1p (Sigma, Cat. # MABC1152; 1:10,000) was used and validated as previously published^{60 (link)}, anti-β-actin is commercially available (Sigma, Cat. #A5316; 1:10,000). Membranes were developed with ECL solution (GE Healthcare) and imaged by Image Lab. The relative expression of the ORF1p protein was measured by densitometry using Image J and normalized for the β-actin intensity of the corresponding lane.