Lumiglo

Muscles powdered in liquid nitrogen were lysed in cold RIPA + buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X, 10% glycerol, phosphatase and protease inhibitors). Subcellular fractionation was done according to Dimauro et al. (2012) [26 (link)]. Following dosage (BCA Protein Assay, Pierce), proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Blots were blocked in TBS, 3% BSA, 0.1% Tween-20, and incubated overnight at 4 °C with primary antibodies, then for 2 h with HRP-labelled secondary antibodies. Immunoreactivity was detected using the Western blot chemiluminescent substrate LumiGLO (Seracare) and exposed to Super RX-N films (Fujifilm) or revealed with iBright™ Imaging System (ThermoFisher). Protein expression was normalized to α-actinin or GAPDH, or to total protein levels of the corresponding phosphorylated form. The list of antibodies used is provided in Supplementary Materials.

Microneutralization antibody titers were determined using RSV-susceptible Vero cells. Heat-inactivated serum samples were serially diluted and mixed with 1000 pfu of the different RSV strains. After 1 h incubation at room temperature, Vero cells were added, and the plates were incubated for 4 days at 37 °C. The monolayers were washed and fixed with 80% cold acetone. RSV replication was determined by F protein expression using a biotin-conjugated anti-F monoclonal antibody MAb8262 (clone 133-1H, Merck, Darmstadt, Germany), which binds to RSV A or RSV B F protein. Plates were incubated with streptavidin-horseradish peroxidase, and after washing, a chemiluminescent substrate (LumiGLO, SeraCare, Milford, MA, USA) was added. The luminescence signal was determined with the Biotek Synergy Neo plate reader. Inhibitory concentration 50% (IC50) titers were calculated.