Modulation of Nrf2 protein levels by GSK-3 is independent of Keap1. (A and C) HEK293T cells were transfected with either V5-tagged Nrf2, the wild type, or the Keap1-insensitive Nrf2 version (Nrf2ΔETGE-V5), maintained in low-serum medium for 16 h, and then treated with 20 μM SB216763 for the indicated times. Upper blots, Nrf2-V5 (A) or Nrf2ΔETGE-V5 (C) protein levels; middle blots, β-catenin levels in the same cell lysates as a control for GSK-3 inhibition; lower blots, β-actin levels showing similar protein loads per lane. (B and D) HEK293T cells were transfected with either Nrf2-V5 (B) or Nrf2ΔETGE-V5 (D). After 24 h they were further transfected with siRNAs for GSK-3α, GSK-3β, or both or with a control scrambled siRNA as explained in Materials and Methods. Cells were lysed 24 h after siRNA transfection. Upper blots, Nrf2-V5 (B) or Nrf2ΔETGE-V5 (D) protein levels; middle blots, GSK-3α and -β protein levels; lower blots, β-actin levels showing that similar amounts of protein were loaded in each lane.