The binding activity of PN-SIA28 was assayed using full-length wild type and mutants HAs cloned as described above. In brief, 1610 6 human epithelial kidney (HEK) 293T cells (ATCC CRL-1573) were transfected in 6 wells plate (Corning, Corning, NY, USA) with 4 mg of pcDNA 3.1D/V5-His-TOPO vector containing the HA nucleotide sequences. After centrifugation and fixation with paraformaldehyde 4%, the transfected cells were incubated for 30 minutes at room temperature with PN-SIA28 or a conformational control for H1N1 (RB62) or a control for H3N2 (12D1, kindly provided by P.Palese) at 1 mg/ml and 10 mg/ml. Additionally, the isotype control, e137 (1 and 10 mg/ml) was introduced as well as untransfected cells and a mouse anti-H1 subtype monoclonal antibody directed against a linear epitope (anti-influenza A hemagglutinin [12D1102] GeneTex Inc., Irvine, CA, USA) to evaluate the transfection efficiency for each HA. The cells were then washed and incubated for 30 minutes at room temperature with FITC-conjugated anti-human (Sigma Aldrich) or anti-mouse (Argene, Shirley, NY, USA) monoclonal antibodies. Afterwards, the cells were washed and analysed by FACS. The FACS data were analyzed using the software Weasel w 2.5 (Waler+Eliza Hall, Institute of Medical Research, Parkville Victoria, Australia). The binding of PN-SIA28 to the different HA-mutants was expressed as a binding percentage compared to wild-type.