Thus, the activity of EBC5-16 is dependent on expression of the EPOR and specific for the human as opposed to the murine version of the receptor. To test whether the transmembrane domain of the hEPOR is required for EBC516 activity, we introduced EBC5-16 into cells expressing an HAtagged hEPOR mutant in which the transmembrane domain of the hEPOR was replaced with that of the murine PDGFbR. We previously showed that BaF3 cells expressing HA-hEPOR proliferated in response to EPO, which binds to the extracellular domain of the receptor retained in the chimera,Benzoylaconine but did not respond to TC2-3 because of the foreign transmembrane domain. As shown in Figure 2D, EBC5-16 also failed to cooperate with HA-hEPOR to induce growth factor independence, indicating that EBC5-16 requires the hEPOR transmembrane domain for activity. To determine whether EBC5-16 causes biochemical activation of the hEPOR, we immunoprecipitated the HA-tagged hEPOR from BaF3/HA-hEPOR cells expressing EBC5-16 or TC2-3 from the low expression vector, RVY-puro, and immunoblotted with an anti-phosphotyrosine antibody. As shown in Figure 3A, EBC5-16 induced tyrosine phosphorylation of the hEPOR. Interestingly, EBC5-16 and TC2-3 induced a similar level of hEPOR tyrosine phosphorylation, despite the enhanced biological activity of EBC516. Similarly,Licochalcone-C EBC5-16 induced tyrosine phosphorylation of JAK2 and STAT5, major downstream signaling partners of the hEPOR, at levels similar to that induced by TC2-3. These experiments demonstrated that we have isolated a TC2-3 mutant with enhanced biological activity in murine cells, but the basis for enhanced signaling has yet to be determined. A fraction of TC2-3 forms a disulfide bond-linked homodimer mediated by the cysteines at the C-terminus of the protein. To determine if EBC5-16 also forms a homodimer, cell extracts were prepared from BaF3/HA-hEPOR cells expressing either EBC5-16 or TC2-3, and replicate samples were immunoprecipitated with aE5, which recognizes the fixed C-terminus of TC2-3 and EBC516.