Dig detection reagents

IFN-β treated HeLa S3 nuclear extract, was pre-incubated for 15 min on ice with 150 fmol linearized template (SpeI/PvuII fragments of basic pGL4[luc2.10], pGL4_OAS1Δ1 or pGL4_mtdupGGAA), in a 20 μl reaction containing, 0.5× binding buffer, 7.5 mM MgCl₂, and 20 U RNAse inhibitor (Toyobo). The reaction was initiated by addition of 0.8 μl of 10 mM rNTP mix containing DIG-11-UTP (Roche Applied Science) to a final concentration of 0.4 mM ATP, 0.4 mM GTP, 0.4 mM CTP, and 0.26 mM DIG-11-UTP. Then incubated at 30 °C for 60 min, stopped with 180 μl stop buffer (10 mM EDTA, 0.2% SDS, 0.3 M sodium acetate, 50 μg/ml yeast tRNA) and RNA-bound proteins digested with 10 μg of proteinase K for 15 min at 55 °C. De novo RNA transcripts were purified by extraction with TE-saturated phenol and then phenol/chloroform, followed by ethanol precipitation. Then, separated by 8 M Urea denaturing PAGE, electrophoretically transferred to a nylon membrane in TBE and cross-linked to the membrane by UV-irradiation. DIG-labeled RNAs were detected with DIG-detection reagents (Roche Applied Science).

Northern blotting was performed as described previously [34 (link)]. A probe named PPYP was used to detect the complementary RNA strand of TLPYFP. The probe corresponds to the 5′ part of the YFP coding region (523 nt) and its upstream 124 nt region of TLPYFP (Fig. 1A). The probe was labeled by incorporating DIG-UTP (Roche Applied Science) during transcription with T7 RNA polymerase. The hybridized probe was detected using DIG detection reagents (Roche Applied Science) and an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).