Horseradish peroxidase conjugated goat anti mouse igg

Concentrated VLPs and infected cell lysates were mixed with SDS solubilizer to final concentrations of 0.0625 M Tris-HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue, 2% (w/v) SDS, and 1% 2-mercaptoethanol. Samples were heated at 95 °C for 5 min, separated in 10% (w/v) polyacrylamide-SDS gels, transferred to PVDF membranes, probed with monoclonal mouse anti-C9 (Santa Cruz) or monoclonal mouse anti-Myc antibody (Santa Cruz, Dallas, TX, USA). Membranes were then probed with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss, Woburn, MA, USA), developed with ECL substrate (Thermo Fisher Scientific, Middlesex, MA, USA), and signals were detected using Fusion Solo X (Vilber, France). Band density on western blot membranes was analyzed using Evolution Capt software (Vilber, France) (shown in Figure S1).

Nanoparticles were incubated with neuraminidase A (Neu A, New England biolabs, Inc., Beverly, MA, USA), neuraminidase S (Neu S, New England biolabs, Inc.), or PNGase F (New England biolabs, Inc.) at 37 °C for 18 h. Proteins were transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). After blocking with 5% (w/v) bovine serum albumin in Tris-buffered saline with 0.05% TWEEN 20 (TBST) for 1 h at 25 °C, membranes were probed with monoclonal mouse anti-flag (1:1000, Sigma-Aldrich, St. Louis, MO, USA) antibodies, biotinylated Sambucus nigra agglutinin (SNA, Vector Laboratories, Burlingame, CA, USA), or biotinylated Maackia amurensis lectin-I (MAL-I, Vector Laboratories, Burlingame, CA, USA) as a primary staining, and then with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss Antibodies, Woburn, MA, USA) and streptavidin at 1:5000 dilution in TBST. Membranes were developed using ECL substrate (Thermo Fisher Scientific, Waltham, MA, USA) and signals were detected with Fusion Solo X (Vilber, Paris, France).

Transfected cells were lysed on ice in Triton X-100 lysis buffer (1% Triton X-100, 50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA) and cleared by centrifugation at 1,000 × g for 10 min at 4°C. Pseudotyped viruses were pelleted by centrifugation at 10,000 × g for 10 h at 4°C. The cell lysates and pelleted pseudotyped viruses were separated in 8% (wt/vol) SDS – PAGE gels, transferred to polyvinylidene difluoride membranes, and probed with monoclonal mouse anti-C9 (EMD Millipore, Burlington, MA, USA) antibody. The membranes were probed with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss, Woburn, MA, USA) and then incubated with enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA). Signals were detected using a Fusion Solo X imaging system (Vilber, Paris, France).