3 end biotinylated rna oligonucleotides

Affinity purification of RNA-binding proteins was performed as previously described [31 (link)], using 3′ end biotinylated RNA oligonucleotides (LGC Biosearch Technologies, Risskov, Denmark): PTS1 (5′-CCAGGUUCGAGCGAUUCUCCAGCCU-3′), PTS1MUT1 (5′-CCAGGUUCGUGCGAUUCUCCAGCCU-3′), PTS1MUT2 (5′-CCAGGUUCUAGCUAUUCUCCAGCCU-3′), PTS1Rev (5′-AGGCUGGAGAAUCGCUCGAACCUGG-3′), PTS5 (5′-UUCUCCAGCCUCAGCCUCCCGAGUA-3′), PTS5MUT (5′-UUCUCCAGCCUCAGCCUCUUGAGUA-3′), PTS5Rev (5′-UACUCGGGAGGCUGAGGCUGGAGAA-3′), PTS6 (5′-UCCCGAGUAGCUGAGAUUACAGGUG-3′), PTS6MUT (5′-UCUUGUGUAGCUUAUAUUUCUGGUG-3′), and PTS6Rev (5′-CACCUGUAAUCUCAGCUACUCGGGA-3′).
Five hundred picomole of each biotinylated RNA oligonucleotide was immobilized in 25 μL Dynabeads M-280 Streptavidin magnetic beads (Invitrogen) and incubated with 293 cell nuclear extract (Protein One). Blank beads were added as a control for background binding. Proteins were eluted in 25 μL protein sample buffer and analyzed by western blotting with antibodies against SRSF1 (32–4500 from Invitrogen), hnRNP A1 (R9778 from Sigma Aldrich), SRSF2 (04-1550 from Millipore), and SR proteins (33–9300 from Invitrogen).