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. 2005 Feb 1;19(3):316–321. doi: 10.1101/gad.319905

Figure 1.

Figure 1.

NQO1 is physically associated with 20S but not 26S proteasomes. (A) Mouse liver extract was precipitated at 38%-70% ammonium sulfate and subjected to Sepharose 6B gel-filtration chromatography. Fractions were collected and analyzed by SDS-PAGE and immunoblotting (IB). (B) Fractions from Sepharose 6B gel-filtration column containing 20S proteasomes were loaded on Resource Q anion-exchange column and eluted with different concentrations of NaCl. Fractions were analyzed by SDS-PAGE and immunoblotting (IB). (C) Purified 26S and 20S proteasomes (0.3 M NaCl fraction) were subjected to nondenaturing PAGE and analyzed for peptidase activity or by immunoblotting (IB). (D) Fractions from Sepharose 6B gel-filtration column containing 20S proteasomes were pooled (Input), and immunoprecipitation experiments were performed with mouse anti-Flag antibody as a control for nonspecific binding (Ab: N.S) or with rabbit anti-C9 antibody (Ab: 20S). The immunoprecipitants (IP) and the supernatant (Sup) were analyzed by SDS-PAGE and immunoblotting (IB) (E) 35S-labeled NQO1 was incubated alone or mixed together with 20S proteasomes (TOTAL). The 20S proteasomes were immunoprecipitated with rabbit anti-C9 antibody (IP: 20S). (F) Fractions from Sepharose 6B gel-filtration column containing 20S proteasomes were pooled and immunoprecipitation experiments were performed with mouse anti-Flag antibody as a control for nonspecific binding (Ab: N.S) or with rabbit anti-C9 antibody (Ab: 20S) in the absence (-) or presence (+) of 200 μM dicoumarol. The immunoprecipitants (IP) and the supernatant (Sup) were analyzed by SDS-PAGE and immunoblotting (IB). Immunoblot analysis (IB) was performed with rabbit anti-C9 antibody to identify the 20S proteasomes, rabbit anti-TBP1 antibody to identify the 26S proteasomes, goat anti-NQO1 antibody, mouse anti-mouse p53 antibody, and with mouse anti-Actin. 35S-labeled NQO1 was detected by autoradiography. Proteasome peptidase activity was determined by their ability to hydrolyze the flurogenic peptide suc-LLVY-AMC, as described in Materials and Methods.