FIGURE 1.
Identification of NTHi Omps interacting with human C4BP. Outer membrane fractions isolated from NTHi 3655 were subjected to two-dimensional SDS-PAGE. The gel was subsequently stained with Coomassie blue for visualization (A). A ligand overlay immunoblotting (far–Western blot) was performed by incubating the membrane with purified human C4BP, sheep anti-human C4BP pAb and HRP-conjugated donkey anti-sheep IgG pAb as a secondary layer (B). Incubation of a membrane without C4BP was also included as negative control to exclude unspecific signals (spots) caused by the Abs (C). The arrow shows Omp P5. The circle indicates multiple isoforms of P5. Binding kinetics of C4BP to P5 loop 2 from NTHi strains 3655 (D) and KR271 (E). The interaction was analyzed by biolayer interferometry (Octet RED96). Purified human C4BP was immobilized on AR2G sensors and the interaction with synthetic P5 peptides were analyzed at different concentrations (1.25–50 µM). Binding affinity (Kd) was calculated by fitting the curves with 1:1 binding kinetics.