Figure 3.

Far Western blot analysis showing C4BP binding to H1N1 and H3N2 proteins. PVDF membrane containing H1N1 and H3N2 virus proteins, separated by SDS-PAGE was blocked with 5% w/v skimmed milk and incubated with 5 µg/ml of C4BP (A) or without C4BP (F) followed by washing steps. The membrane was probed with rabbit-anti-human C4BP polyclonal antibodies (1:1,000) at room temperature for 1 h, followed by washing and incubation with secondary goat anti-rabbit IgG HRP-conjugate (1:1,000) for 1 h at room temperature. Bands corresponding to Hemagglutinin (HA) (~70 kDa), Neuraminidase (NA) (~55 kDa), and Matrix protein 1 (M1) (25 kDa) in the case of both H1N1 and H3N2 subtypes were revealed after developing colour using 3,3′-diaminobenzidine. The identities of C4BP bound IAV glycoproteins were confirmed using separate blots that were directly probed with anti-HA (B), anti-NA (C), anti-M1 (D), or anti-NS1 (E) antibodies.