Inhibition of the TPO-induced mitogenic response by PKC downregulation. (A) Time course for the downregulation of PKC isoforms α and β. UT-7/mpl 5.1 cells were incubated in culture medium lacking GM-CSF, in the absence or presence of 500 nmol/L PDBu, for the indicated lengths of time. At the end of the incubations whole cell lysates were prepared and aliquots equivalent to 0.3 × 10⁶ cells were analyzed by immunoblotting using MoAbs against the indicated PKC isoforms, as described in Materials and Methods. (B) Effect of PKC downregulation on the TPO-induced or GM-CSF-induced mitogenic response. After a pretreatment of 6 hours with 500 nmol/L PDBu, cells were washed three times with culture medium lacking GM-CSF to remove the PDBu, and then incubated for a further 48 hours with 10 ng/mL TPO or 2 ng/mL GM-CSF in culture medium containing 0.1 μCi/mL [³H]thymidine. At the end of this period cells were harvested onto glass fiber filtermats as described in Materials and Methods. Radioactivity values represent the mean ± SD of three replicates. (□), Untreated cells; (▪), cells treated with PDBu.