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. 2012 Apr;32(8):1506–1517. doi: 10.1128/MCB.06271-11

Fig 5.

Fig 5

Competition between PALB2 and NRF2 for KEAP1 binding. (A) Disruption of preformed NRF2/KEAP1 complex in cell extracts. Increasing amounts of whole-cell extracts from 293T cells transfected with pOZC1-PALB2WT or pOZC1-PALB2T92E (lanes 2 and 6, respectively) were added to equal aliquots of extract made from cells cotransfected with HA-NRF2 and Myc-KEAP-GFP. Then, KEAP1 was precipitated using a GFP antibody and the amounts of proteins were analyzed by Western blotting. (B and C) Competitive disruption of isolated NRF2/KEAP1 or PALB2/KEAP1 complexes by IVTed PALB2T551 or NRF2, respectively. See Materials and Methods for detailed procedures. (D) In vitro competition between PALB2 and NRF2 for KEAP1 binding. Different amounts of PALB2 and NRF2 were generated together with a fixed amount of KEAP1 in triple IVT reactions by using different combinations of amounts (in μl) of PCR-generated templates for KEAP1, PALB2, and NRF2 (top panels). Then, KEAP1 was IPed and the amounts of KEAP1, PALB2, and NRF2 in the precipitates were analyzed by Western blotting using anti-HA antibody, which recognizes all 3 proteins (bottom panels).