Figure 5

Isolation of CpG-B ODN-binding proteins from primary salmon mononuclear phagocytes. A, Image of a silver-stained polyacrylamide gel showing ODN-specific, dose-dependent co-precipitation of putative CpG-B ODN-binding proteins. The cells were incubated with the indicated concentrations of biotin-labeled and/or non-biotinylated CpG-B ODNs prior to co-precipitation as described in Figure 1A. The samples were further boiled in LDS sample buffer and subjected to PAGE and silver staining. Addition of an excess of unlabelled CpG-B inhibits the co-precipitation of the bands indicated with “*” suggesting that these represent proteins that specifically bind to the ODNs. B, In order to identify the CpG-binding proteins through MS, milder elution techniques were used either through a combination of biotin and CpGs (left) and CpGs only (right) as described in “Methods”.