Spline curve illustrating interspecific sequence variation across the Tlr4 protein. A multiple alignment of Tlr4 sequences from three rodent species (mouse, rat and hamster) and three primate species (human, chimpanzee and baboon) was generated using the GCG program Pileup. The number of amino acids observed at each residue was plotted using the program Prism 3.0 (a value of 1 was assigned if a single amino acid was observed in the six species; a value of 5 was assigned if five different amino acids were observed among the six species, and so on). The points were then connected using a cubic spline curve. TM, transmembrane domain. Numbering refers to the human sequence. Where a deletion was introduced by Pileup, a single mismatch was assumed. Where the sequence was truncated, each missing residue was tabulated as a separate mismatch.