One set of the samples was then treated with reducing agents to disrupt disulfide bonds, and the other set was left untreated. Samples were then electrophoresed in the presence of SDS to dissociate non-covalent dimers and immunoblotted with aE5. As shown in Figure 5A, under reducing conditions, TC2-3 and EBC5-16 migrated with similar mobility indicative of a monomer. Under non-reducing conditions,Glabridin in addition to the monomeric form, a slower migrating band with mobility expected for a dimer was observed for both proteins, indicating that EBC5-16, like TC2-3, forms a disulfide bond-linked homodimer. Strikingly, a significantly higher fraction of EBC5-16 forms a dimer than TC23. Because there is only a single amino acid difference between the two proteins, this increase in homodimerization is due to the serine residue at position 25. The finding that a large fraction of EBC516 forms a disulfide bond-linked homodimer implies that, like the E5 protein, it adopts a type II transmembrane orientation, placing the C-terminus in the non-reducing extracellular or luminal space. If EBC5-16 interacts directly with the hEPOR, this orientation would align EBC5-16 in an antiparallel fashion relative to the transmembrane domain of the hEPOR, a type I transmembrane protein. In this assay,Licochalcone-A the transmembrane domain to be tested is linked to the monomeric transactivation domain of ToxR, an oligomerization-dependent transcription factor. The level of ToxR-driven chloramphenicol acetyltransferase expression as assessed by measurement of CAT activity is proportional to the strength of oligomer formation induced by the foreign transmembrane segment. To determine if EBC5-16 formed a stronger oligomer than TC2-3, we performed a TOXCAT assay with the transmembrane domains of EBC5-16 and TC2-3 inserted into ToxR. As shown in Figure 5B, the transmembrane domains of TC2-3 and EBC5-16 induced higher CAT activity than the transmembrane domain of the positive control, GpA, indicating that both traptamers form non-covalent oligomers in this system.