IL-4 phosphorylates the region within aa 492-877 of c-Cbl. (A) Constructs of cDNAs for wild-type and deletion mutants of c-Cbl epitope-tagged with an HA peptide. ▿RING-Cbl lacks aa 193-491 including the RING finger domain and ▿PD-Cbl lacks aa 492-877 including the proline-rich domain of c-Cbl. HA, a nine amino acid HA peptide (YPYDVPDYA) from the human influenza virus. RING, the RING finger motif. LZ, the leucine zipper motif. (B) Expression and tyrosine phosphorylation of deletion mutants of c-Cbl by IL-4 stimulation. The cDNAs encoding deletion mutants, ▿RING-Cbl and ▿PD-Cbl, were introduced into Ba/F3 cells by the retroviral vector, and stable transfectants, ▿RING and ▿PD cells were established. These cells were stimulated with IL-4 (10 ng/mL) for 5 minutes at 37°C, lysed, and immunoprecipitated with anti–c-Cbl antibody. The immunoprecipitates were then subjected to the immunoblotting with antiphosphotyrosine antibody (upper panel) or anti-HA monoclonal antibody, 12CA5 (lower panel).