The effect of mutation on NS1 and NS2 expression. (a) Vero cells were infected with wt RSV, NS1F-ELCmut, or mock infected, and cells were lysed in RIPA buffer 24 h and 48 h p.i. Equivalent amounts of protein for each sample were separated by SDS-PAGE and analysed by Western blot using anti-NS1/2 polyclonal antiserum. Representative of three separate experiments. (b) Densitometric quantification of NS1 and NS2 expression in comparison to β-actin for triplicate wt RSV and NS1F-ELCmut-infected Vero cell cultures. NS1F-ELCmut infected cells expressed significantly less NS1 than wt RSV (* P < 0.001). NS2 expression was not different for the 2 viruses. (c) NS1F, NS1F-ELCmut and mock-infected cell lysates were probed with anti-FLAG to detect tagged NS1 protein and anti-RSV polyclonal antiserum to detect other RSV proteins. Representative of three separate experiments. (d) Densitometric quantification of NS1 and N expression in comparison to β-actin for triplicate NS1F and NS1F-ELCmut-infected Vero cell cultures. Cells infected with NS1F-ELCmut expressed significantly less NS1 than cells infected with NS1F (* P < 0.05).