c-Cbl siRNA reduces Tpo-induced c-Mpl ubiquitination and degradation. (A) c-Cbl siRNA significantly reduced expression of c-Cbl in BaF-Mpl cells 48 hours after transfection. (B) c-Mpl ubiquitination was reduced in cells treated with c-Cbl compared with those treated with nontargeting siRNA. The data shown are representative of 4 individual experiments. (C) Tpo-stimulated c-Mpl degradation was analyzed by Western blot using lysates from BaF-WT-Mpl and BaF-MplK553+573R cells treated with c-Cbl–specific and nontargeting siRNAs and pretreated with Chx for 60 minutes before Tpo stimulation for up to 60 minutes. (D) Graphic representation of degradation, comparing the mature (85-kDa) form of c-Mpl in cells treated with c-Cbl–specific siRNA compared with nontargeting siRNA. The data points represent the mean ± SE of 3 independent experiments (*P < .05, **P < .01; Student t test).
