SSL3 has a limited species range and does not inhibit bovine TLR2. A–C Stable HEK293T cell lines expressing human, murine, equine, and bovine TLR2 were stimulated for 6 h with the three TLR2 agonists MALP-2 (A), Pam2CSK4 (B), and Pam3CSK4 (C), in the absence (black) or presence (red) of 10 μg/mL SSL3. Relative IL-8 production was determined by taking the maximum stimulus per species and all data points per species (with and without SSL3) were normalized to this point. Data points represent mean ± SD of two independent experiments. D–F SSL3 binding (final concentrations ranging from 10 to 0.1 μg/mL) to hTLR2, bTLR2, and empty HEK cells was examined without (D) or after neuraminidase treatment (E). HIS-SSL3 was allowed to bind on ice for 30 min before addition of an anti-HIS-FITC monoclonal antibody. Binding was measured using flow cytometry. D, E Histogram overlays of the original flow cytometer data. One representative experiment out of three independent experiments is shown. F Represents the geometric mean fluorescence of the FITC signal (SSL3 binding) of the same binding experiments shown in D and E. SSL3 binding to hTLR2 is shown in black, to bTLR2 in blue, and to empty HEK cells in gray. Solid lines represent the non-treated cell lines and dotted lines represent the cell lines after neuraminidase treatment.