Mutational analysis of the NS5A:FynSH3 domain interaction. The indicated mutations were generated in the Fyn SH3 domain – the numbering refers to the residues in the full length Fyn protein. For reference the Fyn SH3 domain commences at residue 84 . (a) Top panel – Coomassie stained SDS-PAGE of the purified GST-FynSH3 domain fusion proteins. Lower panel – GST pulldown analysis. Purified NS5A(Δ32) was subjected to GST pulldown analysis using GA-beads alone (lane 2), GST (lane 3) or GST-FynSH3 or mutants thereof (lanes 4–11 – corresponding to the GST-FnSH3 mutants in the lanes above). Samples were blotted for NS5A, lane 1 shows 20% of input protein, lanes 2–11 show bound protein eluted by competition with 20 mM reduced glutathione. ELISA analysis. GST-FynSH3 domain fusion proteins were immobilised on microplates and incubated with purified NS5A(Δ32), prior to detection of bound NS5A with sheep anti-NS5A serum. Absorbance values were normalised to the amount of GST fusion proteins loaded (measured by direct ELISA with an anti-GST antibody). The mean value determined over 3 independent experiments is indicated on the graph. (b) Coomassie stained SDS-PAGE of the purified GST-FynSH3 domain fusion proteins and ELISA analysis as in (a).