Schematic diagram of EPO-R variants and their level of expression in 32D cells. (A) Stick figure of the murine EPO-R depicting the various truncated EPO-R isoforms generated (arrows). 1-483 represents the wild-type EPO-R (lacking the signal peptide). The dark box represents the transmembrane domain. The gray boxes represent Box1 and Box2 domains. Specific point mutations constructed are designated. EPO-R(YF) is a full-length EPO-R in which all 8 intracellular (cytoplasmic) tyrosine residues (Y) were changed to phenylalanines. The positions of the tyrosine residues (horizontal line) relative to the sites of truncation are shown. TM, transmembrane domain. (B) Immunoblot analysis of detergent soluble extracts from 32D clones containing the various EPO-R isoforms. All lanes were loaded with a detergent soluble extract from 7.5 × 105 cells. A polyclonal rabbit antisera against the extracellular N-terminal peptide of the murine EPO-R was used. Molecular mass standards (in kilodaltons) are depicted on the right.
