FIG. 7.
Mutation of the 6S cluster within the Neh6 domain renders the Nrf2 protein insensitive to GSK-3. (A) HEK293T cells were transfected with Nrf2-ΔETGE-V5 or Nrf2ΔETGE 6S/6A-V5, maintained in low-serum medium for 16 h, and then incubated with 20 μM SB216763 for the indicated times. Upper blot, anti-V5 immunoblot; middle blot, β-catenin levels as a control of GSK-3 inhibition; lower blot, β-actin levels showing the similar protein loads per lane. (B) HEK293T cells were transfected with either Nrf2ΔETGE-V5 or Nrf2ΔETGE 6S/6A-V5. After 24 h they were further transfected with siRNAs for GSK-3α, GSK-3β, or both or with a control scramble siRNA as described in Materials and Methods. Cells were lysed 24 h after siRNA transfection. Upper blot, anti-V5 antibody; middle blot, GSK-3α and -β protein levels; lower blot, β-actin levels showing similar protein loads per lane.