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Figure 4 | Virology Journal

Figure 4

From: Hepatitis C virus NS5A protein binds the SH3 domain of the Fyn tyrosine kinase with high affinity: mutagenic analysis of residues within the SH3 domain that contribute to the interaction

Figure 4

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 [17]. (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).

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Virology Journal

ISSN: 1743-422X