Responses of human and equine TLR2/1 to synthetic ligands. SW620 cells were transiently transfected with hTLR2 and hTLR1 or eqTLR2 and eqTLR1, together with the cognate species’ CD14 and reporter constructs NF-κB-luc and Renilla luciferase. Cells were stimulated 24 hours later with increasing doses of synthetic ligands Pam2CSK4 and Pam3CSK4. Cells were stimulated for six hours, lysed, and analysed for luciferase activity. Data are from a representative experiment and expressed as triplicate mean ± SEM for that experiment. (A) Pam3CSK4 and Pam2CSK4 stimulated human TLR2/1 dose-dependently, and maximum stimulation by Pam3CSK4 was higher than for Pam2CSK4. (B) Data from (A) were normalised for calculation of EC50, using medium alone as 0% and maxima for each curve (raw data) from (A) as 100%. EC50 values were significantly different for the two ligands, with the Pam2CSK4 curve shifted to the right relative to Pam3CSK4. (C) Pam2CSK4 and Pam3CSK4 stimulated equine TLR2/1 dose-dependently, and maximum stimulation by both ligands was the same. (D) Data from (C) were normalised for calculation of EC50, using medium alone as 0% and maxima for each curve (raw data) from (C) as 100%. The curve for Pam3CSK4 was shifted to the right, and EC50 values were significantly different.