Figure 4

Characterisation of poly(A) signal mutants. A) Poly(A) signal usage after transfection of the indicated plasmids into 293T cells was characterised as described in figure legend 3C. Plasmids pIF^wt Δ2 and pIF^wt Δ24 contain mutations in the second or the second and forth consensus poly(A) signal, respectively. B) Northern blot analysis of cytoplasmic RNA of 293T cells transfected with the indicated plasmids or HEp2 cells infected with RSV. Size separated RNA was stained with ethidiumbromide (EtBr) revealing non-degraded 18S and 28S ribosomal RNA bands (18S shown as representative). A DIG-labelled probe from the RSV-F ORF was used for hybridisation. C, D) Western blot analysis. 293T cells were cotransfected with an EGFP expression plasmid and the indicated RSV-F expression plasmids and analysed by Western blot using an anti-RSV-F monoclonal antibody. The lysate of pIF^syn transfected cells was diluted from 1:10 to 1:10⁴, while the lysates from the other transfected cell were loaded at a 1:1 dilution. Similar transfection efficiencies were controlled for by measuring the fluorescence activity of cell lysates (data not shown).