FIG. 6.
Regulation of Nrf2 by Gα12QL or Gα13QL. (A) Nuclear contents and DNA binding activities of Nrf2. Immunoblot and gel shift assays were performed with the nuclear extracts prepared from cells transfected with empty vector, Gα12QL, or Gα13QL. Cells were treated with 30 μM t-BHQ or 5 μM LPA for 12 h. (B) pGL-1651 reporter assays. Luciferase activity was measured in the lysates of Gα12-deficient cells transfected with pGL-1651 with or without the plasmid encoding Nrf2 or mutant Nrf2 (serine-40 replaced with alanine). Data indicate the means ± standard errors for four separate experiments (asterisks indicate significant differences relative to the respective control [*, P < 0.05; **, P < 0.01]). (C) PKC δ activation. PKC δ was immunoblotted (Western blotted [WB]) in subcellular fractions. (D) Nrf2 phosphorylation. Serine-phosphorylated Nrf2 was immunoblotted with antiserine antibody in Nrf2 immunoprecipitates. Phosphorylation of glycogen synthase kinase 3β was assessed as a positive control for PKC δ activity. IP, immunoprecipitation; PS, phosphoserine. (E) Effects of PKC inhibition on Nrf2 phosphorylation. Serine phosphorylation of Nrf2 was assessed in cells treated with vehicle, PMA (10 μM, 18 h), GF109203X (5 μM, 6 h), or rottlerin (5 μM, 6 h). (F) Role of Gα13 in Nrf2 phosphorylation by Gα12 deficiency. Serine phosphorylation of Nrf2 was determined in the knockdown or knockout cells indicated.