Evolution capt software

The total protein was extracted from the transfected cells using a lysis buffer containing 2% Triton-X, 0.2 mM PMSF, and 1% phosphatase inhibitor cocktail II (Sigma-Aldrich, St. Louis, MO, USA). The protein concentrations were assessed via a Bradford assay using a UV-1700 PharmaSpec spectrophotometer (Shimadzu, Kyoto, Japan) at 595 nM. The samples (20 µg/lane) were then loaded on SDS-PAGE, electrophoresed, incubated with specific primary antibodies (Table S3) overnight at 4 °C, and labeled with HRP-conjugated anti-rabbit or anti-mouse IgG antibodies for 1 h. The blots were developed using a Femto commercial kit (Thermo Fisher Scientific) with a Fusion Solo system (Vilber, Marne-la-Vallée, France). The intensities of the blots were analyzed with Evolution Capt software (Vilber).

For analysis of underglycosylation via densitometry, IgG1 constructs were infiltrated into leaves of N. benthamiana and harvested 3 days post infiltration (dpi). Plant material was mechanically disrupted using a ball mill and 4 µL of ice-cold extraction buffer (0.1 M TRIS, 0.5 M NaCl, 1 mM EDTA, 40 mM ascorbic acid, 2% (w/v), pH 6.8) added to 1 mg of plant material. After removal of cell debris via centrifugation Laemmli buffer was added to the extracts and the samples heated to 95°C for 5 min. Samples were then diluted 1:5 before applying them to SDS-PAGE (12% acrylamide). After electrophoretic separation, proteins were blotted to a nitrocellulose membrane and subsequently detected using an anti-IgG antibody conjugated to horseradish peroxidase (Promega). Proteins were visualized using ECL substrate on a Fusion instrument (Vilber). Densitometric analysis of bands corresponding to glycosylated and unglycosylated IgG1 heavy chain was done using the Evolution-Capt software (Vilber). Background subtraction was done using the rolling ball method of the software.

Total protein was extracted from C2C12 cells using a lysis buffer containing 0.2 mM PMSF, 2% Triton-X, and 1% phosphatase inhibitor cocktail II (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using the Bradford assay with a UV-1700 PharmaSpec spectrophotometer (Shimadzu). The protein samples (20 µg/well) were separated by electrophoresis and transferred onto nitrocellulose membranes (Amersham, Braunschweig, Germany). After blocking with 5% skim milk in TTBS solution (1% Tween 20 in TBS) for 1 h, the membranes were incubated with primary antibodies (Table S3) overnight at 4 °C, washed with TTBS (6 × 5 min), and developed with a secondary antibody (1:10,000 dilution). The blots were then visualized using Femto reagent (Thermo Fisher Scientific, Waltham, MA, USA) and the analytical scanning system Fusion Solo (Vilber, Marne-la-Vallée, France), and the densities of blots were analyzed with Evolution Capt software (Vilber).

For protein preparation, C2C12 cells were lysed and solubilized with PBS containing 2% Triton-X 100 and 0.1% phosphatase inhibitor cocktail, as previously described [53 (link)]. For nuclear/cytoplasmic protein fractionation, the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA) were used by following the manufacturer’s protocol. Proteins (20 μg) were resolved by SDS-PAGE and subjected to immunoblot analysis using specific antibodies (Table S3). Blots were visualized using Femto reagent (Thermo Fisher Scientific), detected by Fusion Solo (Vilber, France), and the intensity of immunoblots was analyzed using Evolution Capt software (Vilber, France).

Briefly, the same titer of pseudovirus expressing spike variants or VSV-G was transduced into three different cell lines (Vero, Caco-2, and Calu-3). At 24 h post-transfection, the cells were harvested with a cell scraper and performed immunoblotting using SARS-CoV-2 spike antibody (Genetex) targeting spike S2 domain. The band intensity of full-length spike and cleaved S2 domain of spike was analyzed by EvolutionCapt software (Vilber Lourmat, Collegien, France) and the ratio of cleaved S2 domain was calculated.

Total cell extracts for Western blot analysis were prepared by trichloroacetic acid (TCA) precipitation from 5-ml aliquots of sporulation cultures, as previously described [27 (link)]. The antibodies used are listed in S3 Table. The ECL, ECL2 or SuperSignal West Femto reagents (ThermoFisher Scientific) were used for detection. The signal was captured on films and/or with a Fusion FX6 system (Vilber) and quantified with the Evolution-Capt software (Vilber).