Figure 3

Transgenic SsTLR9, overexpressed in CHSE cells, spontaneously upregulates ISRE-containing promoters but it does not affect the response to CpGs. A, The cells were cotransfected with the indicated firefly luciferase reporter constructs and either a SsTLR9-expressing plasmid or the empty vector (EV), as indicated. After incubation for 48 hours, to allow for accumulation of transgenic protein, the cells were stimulated for 24 hours with CpG-A, CpG-B and poly(IC). The inducible expression of the firefly luciferase was normalized using the control Renilla luciferase values and is presented as relative luciferase units (RLU). Overexpression of SsTLR9 upregulated the activity of all of the tested promoters except the NFkB reporter. The stimulation of control cells with CpGs and poly(IC) also activated these promoters with variable intensity. The combination of SsTLR9 overexpression and stimulation gave no enhanced response. The error bars show the standard deviation, N = 5, *P < 0.05 vs. EV controls. B, CHSE cells do not express detectable endogenous TLR9 protein. The lysates used for the RGAs were analysed on WB with the SsTLR9 ab. As the antigenic peptide used for the generation of the ab is conserved across Salmo and Oncorhynchus taxa, the ab is expected to recognize the Chinook salmon homolog with similar affinity.