Fluorescence-labeled, double-stranded 100 nM FoxP3 or FoxP3-mut oligonucleotides (FoxP3 forward: 5′-AGCAAAGTTGTTTTTGATAATG-3′, reverse: 5′-CATTATCAAAAACAACTTTGCT-3′; FoxP3-mut forward: 5′-AGCAAAGTTGGGGTTGATAATG-3′, reverse: 3′-CATTATCAACCCCAACTTTGCT-5′; Microsynth, Balgach, Switzerland) [35 (link), 36 (link)] were incubated with 1 µM recombinant FOXO3-DBD protein and CBX in assay buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 5% glycerol) for 30 min at room temperature. For the cell-based FAM-EMSA, 30 µg whole-cell extracts of SH-EP/FOXO3 cells treated with 50 nM 4OHT in combination with CBX were incubated with fluorescence-labeled, double-stranded FoxP3 oligonucleotides (100 nM) in assay buffer for 30 min at room temperature.
The samples were resolved on a 5% polyacrylamide gel in 0.5× TBE running buffer (45 mM Tris-HCL (pH 8.3), 45 mM boric acid, 1.3 mM EDTA). The fluorescence signal was analyzed with the Typhoon 9410 scanner (GE Healthcare, Vienna, Austria).
The samples were resolved on a 5% polyacrylamide gel in 0.5× TBE running buffer (45 mM Tris-HCL (pH 8.3), 45 mM boric acid, 1.3 mM EDTA). The fluorescence signal was analyzed with the Typhoon 9410 scanner (GE Healthcare, Vienna, Austria).
Free full text: Click here
