Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers -- Data Supplement - HTML Page - 07301SuppText.htslp -- Proceedings of the National Academy of Sciences

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.