Supporting Text

Genotyping.

Each mouse embryonic fibroblast (MEF) genotype was determined by PCR with the following primers. For mNrf2 genotyping, the PCR conditions were: 2 min at 96°C, then 20 s at 96°C, 30 s at 59°C, 45 s at 72°C for 35 cycles with primer 3: 5'-TGGACGGGACTATTGAAGGCTG-3'; primer 4: 5'-GCCGCCTTTTCAGTAGATGGAGG-3', which annealed to the mNrf2 gene; and primer 5: 5'-GCGGATTGACCGTAATGGGATAGG-3', which annealed to the lacZ gene located in the knock-in allele. The PCR products of 734-bp and 411-bp bands are derived from wild-type allele and mutant alleles, respectively. For m Keap1 genotyping, the PCR conditions were: 1 min at 94°C, then 30 s at 94°C, 30 s at 60°C, 1 min at 72°C for 30 cycles using primer 6: 5'-CGGGATCCCCATGGAAAGGCTTATTGAGTTC-3'; primer 7: 5'-GAAGTGCATGTAG ATATACTCCC-3', which annealed to the mkeap1 gene; and primer 8: 5'-TCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCA-3', which annealed to the neo gene located in knock-in allele. In this case, the PCR products of 236 bp and 420 bp were derived from wild-type and mutant alleles, respectively.

Constructs.

Each C to A mutant of Keap1 construct was produced by PCR and standard recombination techniques. The following primers were used to introduce the mutations:

For the N-terminal region (NTR): GAACCCAAGCTTAGCGGGGCTCCCCGCAGCAGCCAGTTCCTGCCCCTGTGGTCAAAGgcaCCCGAGGGGGCC; and GCTGAAGGTTCGGTTACCGTCCTGCGAGGGCGTCACCTCTGCCTTagcCTCCGTGGAGGCATACATCAC.

For the C-terminal region (CTR): GGGTCTAGATGCTTCAGCAGGTACAGTTTTGTTGATCAATTTGCTTCCGtgcGGGTTCCATGGTG and GGGTCTAGATGCTTCAggcGGTggcGTTTTGTTGATCAATTTGCTTCCGtgcGGGTTCCATGGTG.

For the intervening region (IVR): GAGGAATTCTTCAACCTGTCACACgctCAGCTGGCCACGCTCATCAGCCGGGATGATCTGAACGTACGCgctGAGTCCGAGGTGTTCCACGCGgcaATCGACTGGGTCAAATACGACgcaCCGCAGCGGCGCTTCTACGTACAGGCACTG; CAGTGCCTGTACGTAGAAGCGCCGCTGCGGtgcGTCGTATTTGACCCAGTCGATtgcCGCGTGGAACACCTCGGACTCagcGCGTACGTTCAGATCATCCCGGCTGATGAGCGTGGCCAG;

GCTTCTACGTACAGGCACTGCTGCGGGCCGTGCGCgctCATGCGCTCACGCCGCGCTTCCTGCAGACGCAGCTGCAGAAGgctGAGATCCTGCAGGCCGACGCGCGCgctAAGGACTACCTGGTGCAGATATTCCAGGAGCTCACGCTGCACAAGCCCAC; GCTTGTGCAGCGTGAGCTCCTGGAATATCTGCACCAGGTAGTCCTTagcGC

GCGCGTCGGCCTGCAGGATCTCagcCTTCTGCAGCTGCGTCTGCAGGAAGCGCGGCGTGAGCGC.

All products were inserted in a subcloning vector and their authenticity was confirmed by sequencing analysis. For mammalian cell expression, they were reinserted into pcDNA3. For producing recombinant protein, C257A-C297A mKeap1 SmaI-XbaI fragment was exchanged with pETmKeap1 SmaI-XbaI wild-type cDNA fragment. Cysteine point mutants of Keap1 were obtained through inserting the appropriate PCR-amplified cDNA fragments into the XbaI and Bam H1 sites of pcDNA3 vector (Invitrogen) for expression in mammalian cells.