Loss and gain of function mutants of human and bovine TLR2. A Bovinized human TLR2 mutants and B humanized bovine TLR2 mutants. Stable HEK293T cell lines were created for all mutant TLR2s (wild-type in red, single mutants in gray, double mutants in green, and triple mutants in blue). Cell lines were stimulated for 6 h with a concentration range of Pam2CSK4 in presence or absence of SSL3. Relative IL-8 production was determined by taking the maximum stimulus for each cell line and all other data points per cell line (with and without SSL3) were normalized to this. C, D Percentage inhibition was calculated at a stimulus of 30 ng/mL Pam2CSK4 for human (C) and bovine (D) mutants. Data points represent the mean ± SD of at least three independent experiments. Statistical significance was determined by a one-way Anova. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001, ns (non-significant), followed by Sidak’s multiple comparisons test. All bars were compared to the control group (human TLR2 for C and bovine TLR2 for D) and if a significant difference was determined for a single mutant, this mutant was subsequently compared to all double or triple mutants.