Nucleotide sequences of the Sp1-like and mutant oligonucleotides used for EMSA
Oligonucleotidea | Sequenceb | Positionc |
|---|---|---|
| Sp D | 5′–GGCGCAGCCCCCTCCCACGGCACCTTTCCA–3′ | -193/-164 |
| Sp Dmut | 5′–GGCGCAGCCCCCTaaaACGGCACCTTTCCA–3′ | |
| Sp C | 5′–AAGATGATGGGAGGGATTTAAAG–3′ | -125/-103 |
| Sp Cmut | 5′–AAGATGATtttAGGGATTTAAAG–3′ | |
| Sp B | 5′–AGGAGGAGGGTGTGGGCAGAAACA–3′ | -72/-49 |
| Sp Bmut | 5′–AGGAGGAtttTGTGGGCAGAAACA–3′ | |
| Sp A | 5′–CCTCTGCCCCACCACCCCCCAAG–3′ | -36/-14 |
| Sp Amut | 5′–CCTCTGCCCCAaaAaCCCCCAAG–3′ | |
| Sp1/3 | 5′–ATTCGATCGGGGCGGGGCGAGC–3′ | Consensus |
↵ a Single-stranded oligonucleotides were synthesized by IDT DNA. Oligonucleotides were resuspended (100 μM). Equal volumes of each oligonucleotide and its antisense oligonucleotide were annealed to form 50 μM double-stranded oligonucleotide, by heating to 95°C and then slowly cooling to RT. One picomole of double-stranded oligonucleotide probe was end-labeled with [γ-32P]ATP using T4 kinase. Sp1/3 is the sequence of a perfect consensus oligonucleotide supplied with the Promega gel shift system
↵ b Putative Sp sites are in bold and underlined. Mutated nucleotides are lowercase
↵ c Position of the oligonucleotides relative to the transcriptional start site