Calf intestine phosphatase

Purified RNA was dephosphorylated using calf intestine phosphatase (Roche). For 5′ end biotinylation, 600 pmol RNA were incubated with 0.2 mM γ-S-ATP (Biomol) and T4 polynucleotide kinase (New England Biolabs) for 30 min at 37°C. Biotin-long-arm maleimide (Vector Laboratories) was added and incubated for 30 min at 65°C. Unincorporated label was depleted by lithium chloride precipitation. Biotinylated RNA (200 pmol) was conjugated to Dynabeads M-280 (Invitrogen) in incubation buffer (10 mM Tris–HCl pH 7.4, 150 mM potassium chloride, 0.5 mM DTT, 0.05% NP40, 100 U/ml RNasin) for 2 h at 4°C with continuous rotation. Whole cell protein lysate (1 mg) of HEK293 cells together with 200 μg yeast tRNA (Sigma-Aldrich) and 5 mg Heparin (Sigma-Aldrich) was added to the beads and incubated 1 h at 4°C followed by 15 min at room temperature with continuous rotation. Beads were washed five times with incubation buffer, resuspended in 30 μl protein loading dye and boiled at 95°C for 10 min. Eluted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad). The primary antibody was anti-AUF1 (1:10 000; rabbit; Millipore; 07-260) and the secondary antibody was Horseradish-conjugated anti-rabbit (1:7000; goat; Jackson ImmunoResearch). Blots were developed using ECL Select (Life Technologies). Imaging was performed on a ChemiDoc Imaging system (Bio-Rad).