Oligonucleotides used here are listed in Table S1. The library expressing randomized mutants of TC2-3 was constructed using a degenerate oligonucleotide mixture in which codons 12 to 30 of TC2-3 were mutagenized, while the remaining codons, 1 to 11 and 31 to 44, remained fixed as TC2-3 sequences. To allow an average of two to three amino substitutions per Dimesna transmembrane domain, the ratio of nucleotides at each mutagenized position was 94% of the wild-type nucleotide from TC2-3 and 2% each of the other three nucleotides. The degenerate oligonucleotide was annealed to a non-degenerate oligonucleotide, which was complementary to the 3�� fixed sequences of the degenerate oligonucleotide and encoded a stop codon and a BamHI restriction site. The oligonucleotides were annealed, extended, and amplified by PCR using short primers complementary to the fixed sequences at the ends of the long oligonucleotides, digested with AvrII and BamHI, and cloned as a mixture of fragments into Paricalcitol pRVY-puro. To gain a better understanding of the structure of small transmembrane activators of the hEPOR and facilitate mechanistic studies, we isolated a more active version of TC2-3. To accomplish this, we first determined whether TC2-3 acts in a cellautonomous fashion or induces the secretion of a soluble factor responsible for growth factor independence. This experiment was conducted in BaF3/hEPOR cells, an IL-3-dependent murine cell line, in which expression of TC2-3 abrogates IL-3 dependence by activating an exogenously expressed hEPOR. A CMMP retrovirus vector with an internal ribosome entry site was used to coexpress TC2-3 and green fluorescent protein from a single transcript in BaF3/hEPOR cells. These cells were co-cultured with an equal number of BaF3/hEPOR cells expressing red fluorescent protein but lacking TC2-3. After growth factor removal and further incubation, the proportion of GFP- and RFPexpressing cells in the culture was assessed by flow cytometry. As shown in Figure 1B, at the time of growth factor removal, the GFP and RFP cells were present in equal number.