FIG. 1.

Nrf2 activation by Gα₁₂ deficiency. (A) Nuclear accumulation of Nrf2 by Gα₁₂ deficiency. Nrf2 was immunoblotted in the nuclear or lysate fractions prepared from knockout cells. Keap1 expression was determined in cell lysates. Graph data indicate the means ± standard errors for three separate experiments (asterisks indicate significant differences relative to RK^−/− MEFs [*, P < 0.05; **, P < 0.01]). (B) Increase in Nrf2 DNA binding by Gα₁₂ deficiency. The arrowhead in the gel shift analysis indicates a Nrf2 DNA binding complex. Each lane contained 10 μg of nuclear extracts and 5 ng of labeled oligonucleotide. An antibody (Ab) competition experiment was carried out by incubating the samples prepared from Gα₁₂-deficient cells with specific antibodies directed against Nrf1 or Nrf2 (2 μg each). (C) Nuclear accumulation of Nrf2 by Gα₁₂ knockdown. For knockdown experiments, RK^−/− cells were transfected with siRNAs directed against Gα₁₂ or Gα₁₃ or nontargeting siRNA. RK^−/− cells were used as a knockout control, which is irrelevant to the function of endogenous G protein in MEFs.