FIGURE 6.

Disruption of NADH binding to NQO1 abolished the interaction between NQO1 and C/EBPα. A, dicumarol disrupts C/EBPα binding with NQO1. HL-60 cells were co-transfected with Flag-C/EBPα and NQO1-V5 and treated with 20 μm dicumarol. Cell lysates were immunoprecipitated and probed with anti-V5, anti-Flag, or anti-20S antibodies. B, mutation of NQO1Y127/Y129 abolished its interaction with C/EBPα. HL-60 cells were co-transfected with Flag-C/EBPα and NQO1(Y127/129A)-V5, cell lysates were immunoprecipitated and immunotblotted. C, NADH binding to NQO1 is essential for NQO1 stabilization of C/EBPα against 20S degradation. In vitro translated C/EBPα was incubated with purified 20S proteasomes in absence and presence of NQO1-V5, NQO1-Y127A/Y129A-V5, NADH, or Dicoumarol for indicated time periods. The incubation mixtures were analyzed by Western blot. D, NQO1P187S mutant is stabilized by V5-tag. HL-60 cells were transfected with hNQO1P187S-V5 or hNQO1P187S plasmids, 24 h later, 10 μm MG132 was included in the media for 10 h. 20 μg cell lysates were loaded for SDS-PAGE, and probed with antibodies as indicated. E, NQO1P187S-V5 stabilizes C/EBPα. In vitro-translated C/EBPα was incubated with NQO1P187S-V5, NADH, and 20S proteasome for different time, incubation mixtures were analyzed by Western blot. All input were 10% of the proteins used in the experiments.