His in the transmembrane domain of c-Mpl is the essential residue for NIP-004. (A) Schema of site-directed mutagenesis in HuMpl and MuMpl. EM indicates extracellular module; TM, transmembrane module; CM, cytoplasmic module. (B) STAT5-reporter gene assay showing induction of STAT activation via HuMpl and MuMpl^L490H but not HuMpl^H499L or MuMpl following NIP-004 administration. Data are expressed as the mean ± SD from 2 independent experiments. (C) Comparison of amino acid sequences of c-Mpl transmembrane domain from various animals. His499 only exists in humans and not in other species.