Figure 5.

Sag regulates the levels of Nrf2 and Erbin. (A and B) Cell lysates were prepared for IB (A), or cells were treated with various concentrations of MLN4924 (MLN), followed by IB (B). (C) Human head and neck carcinoma UMSCC11B cells were either infected with Sag silencing lentivirus or scrambled control, followed by IB. (D) UMSCC11B cells were transiently transfected with Flag-tagged SAG plasmid or the vector control, followed by cycloheximide (CHX) treatment for various time points and were harvested for IB. (E) Human lung H1299 cells were transiently cotransfected with Erbin plus vector control or Erbin plus Flag-tagged SAG, followed by CHX treatment for various time points and harvested for IB. (D and E, top) The data shown are from a single representative experiment out of two repeats. (D and E, bottom) Relative NRF2 and Erbin levels were quantified (n = 2). (F) Cells were treated with CHX for different time periods and subjected to IB. (G) HEK293 cells were transfected with the indicated plasmids for 48 h. Cells were treated with or without MG132 for 3 h, followed by IB. (H) Cells were left untreated or were treated with 10 µM SB216763 for 24 h, followed by IB. (I and J) Cells were transfected with siKeap1 or control siRNA, followed by RT-PCR (I) or IB (J). Error bars indicate the SEM.