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. 2013 May 3;288(25):18393–18405. doi: 10.1074/jbc.M112.438762

FIGURE 11.

FIGURE 11.

Targeted degradation of Nrf2 after phosphorylation by PKC. A, MOCK (GFP shRNA control) or Keap1 knockdown Hep3B cells were transfected with empty pcDNA3.1 vector or pcDNA3.1 containing Myc-S40N or Myc-S40D. Forty eight hours after transfection, the cells were grown under normoxia (Nor) or hypoxia (Hyp) for 6 h. Total cell lysates were immunoblotted using anti-c-Myc, anti-Keap1, anti-HIF-1α, or anti-β-actin antibodies. Values are expressed as the mean ± S.D. of three replicates. *, p < 0.05 significantly different from Myc-S40N alone transfected cells; #, p < 0.05 significantly different from Myc-S40D alone transfected cells. B, MOCK (GFP shRNA control) or Siah2 knockdown Hep3B cells were transfected with empty pcDNA3.1 vector or pcDNA3.1 containing Myc-S40N or Myc-S40D. Forty eight hours after transfection, the cells were grown under normoxia or hypoxia for 6 h. Total cell lysates were immunoblotted using anti-c-Myc, anti-HIF-1α, or anti-β-actin antibodies. Values are expressed as the mean ± S.D. of three replicates; *, p < 0.05 significantly different from Myc-S40D alone transfected cells under normoxia; #, p < 0.05 significantly different from Myc-S40D alone transfected cells under hypoxia. C, MOCK (GFP shRNA control) or Keap1 knockdown Hep3B cells were grown under normoxia or hypoxia for 6 h in the presence of DMSO (vehicle control) or calphostin C (0.1 or 0.2 μm). Total cell lysates were immunoblotted with anti-Nrf2, anti-Keap1, anti-HIF-1α or anti-β-actin antibodies. *, p < 0.05; **, p < 0.01 significantly different from untreated MOCK under normoxia; #, p < 0.05 significantly different from untreated Keap1 knockdown cells under hypoxia. D, MOCK (GFP shRNA control) or Siah2 knockdown Hep3B cells were grown under normoxia or hypoxia for 6 h in the presence of DMSO (vehicle control) or calphostin C (0.1 or 0.2 μm). Total cell lysates were immunoblotted with anti-Nrf2, anti-HIF-1α, or anti-β-actin antibodies. **, p < 0.01 significantly different from untreated MOCK under normoxia; ##, p < 0.01 significantly different from untreated MOCK under hypoxia; *, p < 0.05 significantly different from untreated Siah2 knockdown cells under hypoxia.