Protein g plus protein a agarose beads

Total proteins from vehicle (PBS) or recombinant TSG-6-given pHSCs at 30 min, 1, 2 and 6 h were extracted. To reduce non-specific binding, cell lysates were precleared with Protein G Plus/Protein A agarose beads at 4 °C under rotary agitation for 2 h. Sample were centrifuged at 13,000 r.p.m. for 5 min and supernatants were incubated with mouse anti-His tag primary antibody (sc-8036; Santa Cruz) for detecting recombinant TSG-6 or goat anti-MMP14 primary antibody (af918; R&D Systems) at 4 °C overnight. Protein G Plus/Protein A agarose beads (sc-2003; Santa Cruz) were added to each sample and incubated at 4 °C under rotary agitation for 2 h. Sample were centrifuged at 6000 r.p.m. for 1 min and supernatants were discarded. Beads bounded with protein were washed three times with lysis buffer and boiled in 5 × sample buffer for 10 min. Resulting clear supernatants were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and the following steps were taken for western blot or LC–MS/MS analysis.

To establish a human ACE2 (hACE2)-overexpressing cell line, human ACE2 (NM_021804) cDNA was inserted into the Sgfl/Mlul site of the pCMV6-GFP expression vector plasmid (OriGene Technologies, Inc., Rockville, MD, USA). The resulting pCMV6-ACE2-GFP was then transfected into HepG2 cells (ATCC HB-8065) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were cultured in Opti-MEM (Invitrogen, Carlsbad, CA, USA) at 37 °C for 5 h and then placed in freshly changed culture medium. The diluted sera from vaccinated mice were mixed with SARS-CoV-2 pseudovirus and incubated at 37 °C for 1 h before adding to hACE2-transfected HepG2 cells in 48-well culture plate for a 24-h incubation period in DMEM (Invitrogen, Carlsbad, CA, USA) at 37 °C, 5% CO₂. To detect S-protein immunoprecipitated by hACE, hACE of cell lysate was first immunoprecipitated by hACE monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) with Protein G plus/Protein A agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Thereafter, the precipitates were blotted with monoclonal anti-S-protein antibody (Sigma-Aldrich, St. Louis, MO, USA).