Sequence and conditions of oligos used for PCR amplification and EMSA
Primer Name | Sequence (5′-3′) | Conditions | |
|---|---|---|---|
| Oligos for amplifying the cDNA | |||
| CAR-F1 | GGAGAGGCATTCCATACCAG | 94°C × 30 s, 58°C × 30 s, 72°C × 90 s, 32 cycles | |
| CAR-R1 | TTCCCACTCCAGTGTATCCAG | ||
| CAR-F2 | CAGGTGACATGCTGCCTAAG | 94°C × 30 s, 57.3°C × 30 s, 72°C × 90 s, 15 cycles | |
| CAR-R2 | TATCCAGGGTGTTCCAGGTG | ||
| CAR-Taq-F | CCAGCTCATCTGTTCATCCA | 94°C × 30 s, 57°C × 30 s, 72°C × 30 s, 30 cycles | |
| CAR-Taq-R | GGTAACTCCAGGTCGGTCAG | ||
| CYP2B6-F | CACTCATCAGCTCTGTATTCG | 94°C × 30 s, 57°C × 30 s, 72°C × 30 s, 30 cycles | |
| CYP2B6-R | GTAGACTCTCTCTGCAACATGAG | ||
| MRP4-F | GCAAGATGCTGCCCGTGTAC | 94°C × 30 s, 60°C × 30 s, 72°C × 30 s, 30 cycles | |
| MRP4-R | ACCCTTGCAACTCCTCTCCAAG | ||
| Oligos for EMSA | |||
| CYP2B6-NR1 | GATCCTGTACTTTCCTGACCCTGAGATC | ||
| CYP2B6-NR2 | GATCCATGGACTTTCCTGAACCAAGATC | ||
| Oligos used for creating ATG mutants | |||
| CAR-ATG-exon1-220-F | GTGGCCTGCAGGTGACTTGCTGCCTAAGAGAAGC* | 95°C × 30 s, 55°C × 30 s, 68°C × 12 min, 12 cycles | |
| CAR-ATG-exon-220-R | GCTTCTCTTAGGCAGCAAGTCACCTGCAGGCCAC* | ||
| CAR-ATG-exon4-645-F | CCCACACCCGCCACTTGGGCACCATGTTT* | 95°C × 30 s, 55°C × 30 s, 68°C × 12 min, 12 cycles | |
| CAR-ATG-exon4-645-R | AAACATGGTGCCCAAGTGGCGGGTGTGGG* | ||
| CAR-ATG-exon-654-F | GCCACATGGGCACCTTGTTTGAACAGTTTGTG* | 95°C × 30 s, 55°C × 30 s, 68°C × 12 min, 12 cycles | |
| CAR-ATG-exon-654-R | CACAAACTGTTCAAACAAGGTGCCCATGTGGC* | ||
| Primers used to amplify the cDNA from hepatocytes | |||
| CYP3A4-F | CCAAGCTATGCTCTTCACCG | 95°C × 30 s, 50°C × 30 s, 72°C × 30 s, 28 cycles | |
| CYP3A4-R | TCAGGCTCCACTTACGGTGC | ||
| CYP2B6-F | TCCTTTCTGAGGTTCCGAGA | 95°C × 30 s, 58°C × 30 s, 72°C × 30 s, 35 cycles | |
| CYP2B6-R | TCCCGAAGTCCCTCATAGTG | ||
| PXR-F | CAAGCGGAAGAAAAGTGAACG | 95°C × 30 s, 60°C × 30 s, 72°C × 30 s, 28 cycles | |
| PXR-R | CTGGTCCTCGATGGGCAAGTC | ||
| GAPDH-F | ACCACAGTCCATGCCATCAC | 95°C × 30 s, 60°C × 30 s, 72°C × 30 s, 25 cycles | |
| GAPDH-R | TCCACCACCCTGTTGCTGTA |
| |
↵* Bolded nucleotide indicates the base mutated to change ATG to TTG.