Figure 2.

NQO1 selectively protects p73α, but not p73β, from 20S proteasomal degradation. (A) ³⁵S-labeled p53 was incubated without (-) or with (+) 20S proteasomes at 37°C for 15, 30, and 60 min, without (-) or with (+) 50 μM MG132. (B) ³⁵S-labeled p73α was incubated without (-) or with (+) 20S proteasomes without (-) or with (+) 50 μM MG132 at 37°C for 1 h. (C) ³⁵S-labeled Flag-p73α or immunoaffinity-purified ³⁵S-labeled Flag-p73α were incubated without (-) or with (+) 20S proteasomes at 37°C for 1 h. (D) ³⁵S-labeled p53 was incubated in the presence of 1 mM NADH without (-) or with (+) 20S proteasomes, without (-) or with (+) ³⁵S-labeled NQO1 (E) ³⁵S-labeled p73α or p73β were incubated in the presence of 1 mM NADH without (-) or with (+) 20S proteasomes, without (-) or with (+) ³⁵S-labeled NQO1. ³⁵S-labeled p73 and NQO1 were analyzed by SDS-PAGE and detected by autoradiography. The 20S proteasomes were detected by immunoblot analysis (IB) with rabbit anti C9 antibody. (F) Cell extracts of HCT116 cells and HCT116 cells that stably express pEFIRES NQO1 were fractionated by SDS-PAGE; immunoblot analysis (IB) was carried out with mouse anti-p53, with rabbit anti-p73 antibody, and with goat anti-NQO1 antibody.