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. 2004 Oct;24(19):8477–8486. doi: 10.1128/MCB.24.19.8477-8486.2004

FIG. 3.

FIG. 3.

Inactivation of Cul3 stabilizes Nrf2. (a) 293T cells were transfected with increasing concentrations of plasmids encoding Myc-Keap1 (lanes 1 to 5) or Flag-Cul3 (lanes 6 to 10). The expression of Nrf2, Myc-Keap1, and Flag-Cul3 was assessed via immunoblot. (b) 293T cells were mock transfected (lanes 1 and 2) or transfected with a plasmid encoding the N-terminal 418 residues of Cul3 (lanes 3 to 5) and treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 4 h. Total Nrf2 levels were detected via immunoprecipitation followed by immunoblot analysis, and total Cul3 levels were determined by immunoblot analysis. Lane 5 shows a control precipitation with a nonspecific antiserum. (c) 293T cells were mock transfected (lane 1) or transfected with plasmids expressing short hairpin RNAs against firefly luciferase (lane 2) or Cul3 (lanes 3 and 4). Total Nrf2 levels were detected by immunoprecipitation followed by immunoblot analysis. Total Cul3 and β-tubulin levels were determined via immunoblot analysis. Lane 5 shows a control precipitation with a nonspecific antiserum. (d) 293T cells transfected with plasmids encoding HA-Nrf2 and Myc-Keap1 in the absence (lanes 2 to 6) or presence of a plasmid encoding the N-terminal 418 residues of Cul3 (lanes 7 to 11) were pulsed with [35S]methionine followed by the addition of complete methionine-containing medium for the indicated intervals. Nrf2 was immunoprecipitated from whole-cell lysates, and proteins were resolved via SDS-PAGE and visualized by autoradiography. Lane 1 shows a control precipitation with a nonspecific antiserum. (e) 293T cells transfected with plasmids encoding HA-Nrf2 and Myc-Keap1 in the absence (lanes 2 to 5) or presence of a plasmid encoding short hairpin RNA against Cul3 (lanes 6 to 9) were pulsed with [35S]methionine followed by the addition of complete, methionine-containing medium for the indicated intervals. Nrf2 was precipitated from whole-cell lysates, and proteins were resolved via SDS-PAGE and visualized via phosphorimaging. Lane 1 shows a control precipitation with a nonspecific antiserum. (f) 293T cells were transfected with plasmids encoding short hairpin RNAs against firefly luciferase (lane 2) or Keap1 (lanes 3 and 4). Total Nrf2 levels were determined via immunoprecipitation followed by immunoblot analysis.