Table 1. Oligonucleotides used in this study.
| Experiment | Location# | |
|---|---|---|
| EMSA≠ | • CRE oligo 5′-TTCCTGCGATTCAATGACATCACGGCTGTG-3’ • Mutant CRE oligo 5’TTCCTGCGATTCAGAATTAGTGCGGCTGTG 3’ |
-127 to -97 |
| • Sp1 oligo 5’GCCACCGACCCCACCCCCTGCCTGGA-3’ • Mutant Sp1 oligo 5’GCCACCGTCTTGATCTCCTGCCTGGA3’ |
+53 to +79 | |
| DNA methylation analysis | Outer | |
| • t-PA prom 10 F 5’TTTGAAAAGGTGTTAGTAAG3’ • t-PA prom 10 R 5’ACCACTAAAAAAACAAAACC3’ |
-721 to -382 | |
| • t-PA prom12F2 5’GTTATTATAGGGTTTTGAAAG3’ • t-PA prom12R 5’AAAAAAACAAACCCCAAAATACAA3’ |
-217 to +220 | |
| • t-PA prom 14 F 5’TTTGGGTTTATTTAAGGGGATGT3’ • t-PA prom14R 5’AAAAATTTTCTCTCCAACCCTAAAC3’ |
-577 to +156 | |
| • t-PA enh 23 F 5’GAGAGAGGAGTTATGGAAAG3’ • t-PA enh 23 R 5’TACATATAATCCCAACTACT3’ |
-7538 to -6992 | |
| • t-PA enh27F 5’ATTATTGTATTTTAGGTAGGGTG3’ • t-PA enh27R 5’CCTTCCTAAATCAAACATTTTT3’ |
-8118 to -7470 | |
| Inner | ||
| • t-PA prom11F 5’TAAGGGAAATGGTTTGTTTA 3’ • t-PA prom 11 R 5’CTACRATAAAAAATACCCCCATA3’ |
-704 to -416 | |
| • t-PA prom 13 F3 5’TTTTTAAGTTTGGGATATTAGGA3’ • t-PA prom13R 5’AAAATTTTCTCTCCAACCCTAAACT3’ |
-193 to +155 | |
| • t-PA prom15 F5’GAGGTTATTTATTGTAGTTTTGTATTTTAT3’ • t-PA prom15 R 5’CAACTCTAAACTCCCCACAACTC 3’ |
-535 to +118 | |
| • t-PA enh 23F 5’GAGAGAGGAGTTATGGAAAG3’ • t-PA enh 22R2 5’ATCCCAAACCATAACTATAT3’ |
-7538 to -7216 | |
| • t-PA enh 21F 5’AGTTTTTGTTGTGGAAGTTA3’ • t-PA enh 21 R2 5’ATAAAAAAATCCCTTAAACC 3’ |
-7353 to -7024 | |
| • t-PA enh28F 5’GTGTTTTTTTATTTGATTTATGTT3’ • t-PA enh28R 5’ ATAACTTTCCATAACTCCTCTCT3’ |
-8048 to -7515 | |
| RACE | • PLATgSp1R 5’CACACAGCAGCAGCACACAGCAGAGCCCTC 3’ • PLATgSp1innR 5’CAGAGCCCTCTCTTCATTGC3’ • Forward primers were provided with the 5’ RACE kit (Clontech) biotin-AGGGACGCTGTGAAGCAATCATGG |
• -254 • -235 • -213 |
| Reporter gene | t-PA prom 26 F 5’GTTTATGTGAGCAAACAGCAGA 3’ | |
| Real time qPCR | • t-PA-F 5’ CCGGCTACGGCAAGCA 3’ • t-PA–R 5 ‘AGCGGCTGGATGGGTACA’3’ • EAR-F 5’-GAGGCTGAGGCAGGAGAATCG-3’ • EAR-R 5’-GTCGCCCAGGCTGGAGTG-3’ |
# The location indicates the position of the 5’ end of the oligo with respect to TSS1 of the t-PA gene or, for 5’RACE analysis, with respect to the mRNA sequence for a transcript starting at TSS1.
NB. F and R denote forward and reverse primers, respectively.
≠ For the electrophoretic mobility shift analysis (EMSA) only the forward oligo sequence is given. Underlined are the CRE or Sp1 core recognition sites. In bold the CpGs near the core sequences.
For RACE analysis the location is given with respect to TSS1. EAR = The expressed Alu repeat