Fig 6. Western Blot analysis of NRF2 levels in selected screening hits.
(A) Anti-NRF2 Western Blot of tissue cell lysates after siRNA to the indicated screening hits. Treatment of cells with MLN4924, a small molecule inhibitor of CUL3 and other Cullin-RING ligases, leads to a strong increase in NRF2 levels as compared to control siRNA cells (CTRL). siRNA downregulation of CUL3, KEAP1, PSMC2, PSMC4, PSMD6 and RAB28 also lead to increases in NRF2 levels. Poly-ubiquitylated species of NRF2 after downregulation of proteasomal subunits are also apparent (NRF2ub). A Western Blot against Actin is included as loading control. All samples shown were run on the same gel. Dashed lines indicate where lanes have been removed for clarity. (B) Quantification of NRF2 Western Blots for the indicated siRNA samples relative to siRNA control samples. Asterisk indicates significant stabilization of NRF2 (p<0.05).