SsTLR9 binds PS-modified ODNs through a CpG-independent but a pH-dependent mechanism. A, Specific binding of CpG-B ODNs to SsTLR9. To up-regulate the SsTLR9 protein, primary salmon mononuclear phagocytes were pretreated with IFN-γ for 48 hours, as indicated. The cells were further stimulated with either biotinylated “+” or non-biotinylated “-” CpG-B (2 μM) for 2 hours. To further confirm the specificity of the binding, a sample was treated with biotinylated CpG-B in the presence of 10 μM non-biotinylated CpG-B (indicated with “+/–”). Following the stimulations, cell lysates were subjected to a pull-down analysis with streptavidin-conjugated magnetic beads and then analyzed on WB with SsTLR9 ab. In addition, the whole lysates were analyzed in parallel to confirm the SsTLR9 up-regulation and loading. In the pull-down blot, the SsTLR9 signal (~120 kDa) is detectable only in the samples stimulated with biotinylated CpG ODNs. The addition of an excess (5-fold higher concentration) of non-biotinylated ODNs significantly reduces the amount of the co-precipitated SsTLR9 which further confirms the specificity of the binding of the receptor to the ODNs. B, The interaction between ODNs and SsTLR9 is enhanced under low pH conditions. The pull-down was performed under the conditions described above and the pH of the lysis buffer was adjusted to the indicated values using 3 M HCl. In addition, inverted non-stimulatory CpG-B (ODN 2007 T) was included in the experiment. The results demonstrate that SsTLR9 binds to both the CpG and the inverted CpG ODNs with similar avidity which was highest at pH 5.5. The results shown in both panels were confirmed in at least two independent experiments.