Expression of transfected wild-type and chimeric receptors but not of endogenous EpoR, GMR-α and IL-5Rα in Ba/F3 clones. (A) Surface expression of EpoR and E/β receptors (left-hand side) or IL-5Rα and IL-5α/βIL3 receptors (right-hand side). 2 × 105 cells were treated as described in Materials and Methods and processed for FACS analysis. The type of cells and the antibodies used are indicated in each panel. Anti-EpoR antibody: antibody 8866; isotype control 1: polyclonal rabbit antimouse. Anti-IL-5Rα antibody: antibody 20H9; isotype control 2: polyclonal rat antimouse IgG2b. A representative sample of 3 independent experiments is shown. (B) Detection of 3′ specific EpoR, GMR-α, and IL-5Rα sequences, respectively. Twenty-five micrograms of total RNA was analyzed in each lane. Probes are described in Materials and Methods. Migration of each protected fragment agrees with the expected size and is indicated by an arrow. Top panel: RNase protection assay for EpoR RNA sequences. M: DNA markers (pBR322 Msp I digest); t: tRNA. WT: Ba/F3 cells; EpoR: Ba/F3-EpoR cells; E/β: Ba/F3-E/β cells. Middle panel: RNase protection assay for GMR-α RNA sequences. t: tRNA; WT: Ba/F3 cells; GMR-α: Ba/F3-GMR-α cells; GMER: Ba/F3-GMER cells; GMα/βIL3 : Ba/F3-GMα/βIL3 . Bottom panel: RNase protection assay for IL-5Rα RNA sequences. M; DNA markers; t: tRNA; WT: Ba/F3 cells; IL-5Rα: Ba/F3-IL-5Rα; IL-5α/βIL3 : Ba/F3-IL-5α/βIL3 .
