FIGURE 4.

Fbw7-mediated degradation of GATA2 is Thr¹⁷⁶-dependent. A, effects of Fbw7 on GATA2 stability. HeLa cells were transfected with WT-GATA2-myc-His or T176A-GATA2-myc-His in the absence or presence of Fbw7 or ΔF-box mutant (ΔF). After 41 h of transfection, cells were treated with or without 10 μm MG132 for 7 h and then harvested. Total cell lysates were subjected to immunoblotting with the indicated antibodies. B and C, effects of Fbw7 on GATA3 and GATA1 stability. HeLa cells were transfected with GATA3-myc-His or FLAG-GATA1 in the absence or presence of HA-Fbw7 and then were treated with or without 10 μm MG132 for 7 h. Levels of GATA3 (B) and GATA1 (C) were analyzed by immunoblotting with the indicated antibodies. D, effects of ectopic expression of Fbw7 on degradation of WT-GATA2 or T176A-GATA2. HeLa cells were transfected with WT-GATA2-myc-His in the absence or presence of FLAG-Fbw7, treated with 20 μg/ml CHX for the indicated periods, and harvested. Total cell lysates were subjected to immunoblotting. The representative data of WT-GATA2 are indicated (D). E and F, levels of WT-GATA2 and T176A-GATA2 in the absence or presence of FLAG-Fbw7 after the various chase times were quantitated by image analysis and normalized against β-actin. The percentages of remaining GATA2 protein were calculated as the mean ± S.D. (error bars) from three independent experiments. The p value was determined by Student's t test. *, p < 0.05; **, p < 0.01.