FIG. 3.

Ubiquitination of Nrf2, mediated by the Keap1-Cul3-Rbx1 E3 ubiquitin ligase, occurs in the cytosol. (A) NIH 3T3 cells were singly transfected with an expression vector for Flag-Cul3 (upper panel), cotransected with expression vectors for both Flag-Cul3 and Keap1 (middle panel), or cotransfected with expression vectors for Flag-Cul3, Keap1, and HA-Nrf2 (lower panel). Subcellular localization of Cul3, Nrf2, or Keap1 was determined by indirect immunofluorescence analysis using anti-Flag for Cul3 (panels A, D, G, K, O, and T), anti-HA for Nrf2 (panels P and U), or anti-Keap1 for Keap1 (panels H, L, Q, and V). Nontreat, nontreated; LMB, LMB treatment. (B) Subcellular distribution of Cul3, Nrf2, and Keap1 in singly transfected cells in the absence or presence of LMB was determined by indirect immunofluorescence staining. At least 100 positive cells were examined. Percentages of cells that localized predominantly in the cytosol (C), the whole cell (W), or the nucleus (N) were presented as a bar graph. K-WT, Keap1-WT; N-WT, Nrf2-WT; +LMB, LMB treatment. (C) MDA-MB-231 cells were cotransfected with expression vectors for the indicted Nrf2 and Keap1 proteins. Fifty micrograms per milliliter cycloheximide was added 36 h after transfection. Total cell lysates were collected at the indicated time points following cycloheximide treatment and subjected to immunoblot analysis with anti-HA antibodies. (D) The relative intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system from Bio-Rad and plotted on a semilog scale. The amount of Nrf2 present at the beginning of cycloheximide treatment was set at 1. The half-life of Nrf2 in each group was indicated.