Imagequant tl 8.2 image analysis

Proteins in 20 × PBS-diluted serum samples were separated by 8–16% gradient SDS-PAGE precast gels (Bio-Rad, Hercules, CA, USA). To preserve oligomeric states of A-SAA during SDS-PAGE, the sample buffer did not contain β-mercaptoethanol and the samples were not boiled. A-SAA proteins were then recognized by an anti A-SAA antibody (ab687, Abcam) and a secondary antibody (goat anti-mouse IgG peroxidase conjugated antibody, Jackson ImmunoResearch Labs, West Grove, PA, USA) and visualized in a luminescence/fluorescence imaging system ImageQuant LAS 4000 biomolecular imager (GE Healthcare, Marlborough, MA, USA). The amount of A-SAA monomer and oligomer was quantified by ImageQuant TL 8.2 image analysis (GE Healthcare). The final results were calculated by normalization of the amount of A-SAA monomer and oligomer from each patient to the mean amount of the hepatitis group.

Fusion assays were performed as described before (Mykytyn et al., 2021 (link)). Briefly, HEK-293T cells were transfected with 1.5 μg pGAGGS-spike (all coronavirus S variants described above) DNA and pGAGGS-β-Actin-P2A-7xGFP11-BFP DNA or empty vector DNA with PEI in a ratio of 1:3 (DNA: PEI). Transfected HEK-293T cells were incubated overnight at 37°C 5% CO₂, resuspended in PBS and added to GFP1-10 expressing VeroE6, VeroE6-TMPRSS2 and Calu-3 cells in Opti-MEM I (1X) + GlutaMAX at a ratio of 1:80 (HEK-293T cells: GFP1-10 expressing cells). Fusion events were quantified by detecting GFP+ pixels after 18 hr incubation at 37°C 5% CO₂ using Amersham Typhoon Biomolecular Imager (channel Cy2; resolution 10 µm; GE Healthcare). Data was analyzed using the ImageQuant TL 8.2 image analysis software (GE Healthcare) by calculating the sum of all GFP+ pixels per well.