Clx imaging device

For preparation of protein extracts, HBEpC were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 8.0, 1% (v/v) Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.3% (v/v) SDS, 5 mM NaF, 1 mM Na₃VO₄, 1x cOmplete Mini, EDTA-free protease inhibitor cocktail (Merck), 2 mM MgCl₂ and 1 U/mL Pierce^TM Universal Nuclease (Thermo Scientific). Protein concentrations were determined using DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) samples were separated by SDS-PAGE and were blotted onto Amersham^TM Protran^TM Premium 0.45 µm nitrocellulose membrane (Cytiva, Marlborough, MA, USA) for 1.5 h using const. 120 V. Membranes were blocked for 1 h in 5% (w/v) BSA in TBS-t (150 mM NaCl, 20 mM Tris, 0.1% (v/v) Tween-20) and incubated with SARS-CoV-2 (2019-nCoV) Spike S1 Antibody (1:1000, #40150-R007, Sino Biological, Beijing, China), SARS-CoV/SARS-CoV-2 Nucleocapsid antibody (1:1000, #40143-MM05, Sino Biological, Beijing, China) and β-actin antibody (1:1000, #4967, Cell Signaling Technology, Leiden, The Netherlands) overnight at 4 °C in blocking buffer. Secondary antibodies IRDye^® 800CW goat anti-rabbit IgG (H + L) and IRDye^® 680RD goat anti-mouse IgG (H + L) (LI-COR Biosciences GmbH, Bad Homburg, Germany) were diluted 1:40.000 in blocking buffer and incubated for 1 h at room temperature. Blots were imaged using CLx imaging device (LI-COR Biosciences GmbH, Bad Homburg, Germany).

For preparation of protein extracts, cells were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 8.0, 1% (v/v) Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.3% (v/v) SDS, 2 mM MgCl₂). Buffer was supplemented with 5 mM NaF, 1 mM Na₃VO₄, 20 mM β-glycerophosphate, 1x cOmplete Mini, EDTA-free protease inhibitor cocktail (Merck KgaA; Darmstadt, Germany) and 1 U/mL Pierce^TM Universal Nuclease (Thermo Fisher; Waltham, Massachusetts, United States). Lysates were stored at −20°C. Protein concentrations were determined using DC Protein Assay Kit (Bio-Rad; Hercules, CA, United States). Proteins were separated by SDS-PAGE and were blotted onto an Amersham™ Protran® nitrocellulose membrane (Merck KgaA; Darmstadt, Germany) for 1.5 h using const. 120 V. Blots were washed and blocked for at least 1 h using 5% (w/v) BSA/TBS-t (150 mM NaCl, 20 mM Tris, 0.1% (v/v) Tween20). Primary antibody (Supplementary Table S11) incubation was performed overnight at 4°C in 5% BSA-TBS-t. After washing, secondary antibody was incubated for 1 h in 5% BSA-TBS-t. Blots were imaged using CLx imaging device (LI-COR; Lincoln, Nebraska, United States). All full-length western blots are presented in Supplementary Figure S5.