Involvement of Shc in IL-2–mediated lineage conversion in CLPs. (A) CTLL-2 transfectants were stimulated with cytokines as indicated in the figure. Cell lysates were subjected to SDS-PAGE or immunoprecipitation. Immunoprecipitates were resolved by SDS-PAGE and transferred to membranes. Membranes were probed with anti-pTyr and anti-Shc antibodies. Whole-cell lysates (WCL) were used for the detection of pERK1/2 and ERK2. (B) Schematic depiction of IL-2Rβ mutants. The d325 lacks both A and H regions. A full-length Shc is fused to the C terminal of the d325 mutant, resulting in d325-Shc. (C) IL-2Rβ mutants were introduced into CLPs by retroviral transduction. GFP⁺ cells were sorted and cultured on OP9 stromal layers in the presence of IL-3 and GM-CSF. At day 5 of culture, cells were harvested and subjected to FACS analysis. Percentages indicate total cells in the gates as described in Figure 1C.