The effects of c-Cbl in IL-4R signaling. (A) Establishment of HCbl cells that overexpress c-Cbl. Lysates from HCbl and mock cells were subjected to the immunoblotting with anti–c-Cbl antibody (upper panel) and anti-p85 antibody (lower panel). Arrows indicate c-Cbl and p85. We think that bands located below p85 are nonspecific bands. (B) Overexpression of c-Cbl enhances transient growth and elongates survival of Ba/F3 cells in the presence of IL-4. HCbl and mock cells were cultured in RPMI containing 5% FCS and IL-4 (1 ng/mL) in the presence (+WT, 50 nmol/L) or absence of wortmannin. (C) Survival effects of IL-4 enhanced by c-Cbl is sensitive to wortmannin. Low molecular weight DNA extracts from HCbl and mock cells cultured with or without IL-4 (1 ng/mL) plus wortmannin (WT, 50 nmol/L) for 48 hours were electrophoresed on an agarose gel (2%). (D) HCbl and mock cells were deprived of IL-3 for 24 hours. IL-4 (1 ng/mL) and wortmannin (WT, 50 nmol/L) were then added to the medium, incubated for 12 hours, and subjected to the [³H]thymidine incorporation assay. The growth rate was expressed as a ratio over the basal values at no IL-4 stimulation.