Ab20745

At the indicated time points cells were lysed (25 mM glycerol phosphate, 20 mM tris, 150 mM NaCl, 1 mM EDTA, 1% triton X-100, 10% glycerol, 50 mM NaF, 5 mM Na₄O₇P₂, pH 7.4), and western blotting was performed^{19 (link),43 (link)}. Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF; Millipore) membranes. After blocking in 10% (w/v) milk in TBS-T, the appropriate primary antibodies were added at 4 °C overnight: sheep α-BUNV-N (1:5000) produced by J N Barr, University of Leeds (antibody-containing serum was obtained from a sheep inoculated with BUNV-N protein previously expressed in E. coli and purified by affinity and size-exclusion chromatography detailed in Ariza et al. (2013)^{44 (link)}; and used extensively in previous publications^{18 (link)}); goat α-HRSV (1:1000; Abcam ab20745); mouse α-GAPDH (1:2000, Santa Cruz sc47724); or mouse α-GFP (1:1000; Santa Cruz sc-9996). This was followed by the corresponding HRP-conjugated secondary antibodies (Merck α-sheep A3415; α-goat A8919; α-mouse A4416) for 1 hr. Labelling was detected using enhanced chemiluminescence (Advansta) and a G:Box processor. Densitometry was performed using ImageJ, showing standard deviation error bars and statistical significance determined using a one-way ANOVA where P < 0.05. Full uncropped blots and densitometry data can be found in the Source Data file.