Bas ip imaging plate

In vitro translation was performed using a TnT (transcription/translation)-coupled rabbit reticulocyte lysate system (Promega). Total reaction volumes of 10 μl containing luciferase expression plasmid DNA, rabbit reticulocyte lysate, [³⁵S]methionine, TnT reaction buffer, T7 RNA polymerase, amino acid mixture (-Met), RNase inhibitor and increasing amounts of RNAs were incubated for 90 min at 30°C according to the manufacturer’s recommendations. Proteins in reaction mixtures were resolved by SDS-PAGE. Gels were then dried, placed on the BAS-IP imaging plate (Fujifilm), and analyzed on an FLA-7000 phosphor-image analyzer (Fujifilm).

In vitro-transcribed BC200 RNA was renatured as described (Jung et al., 2014 (link)). BC200 RNA-eIF4A complex formation was performed in 100 μl of phosphate-buffered saline (PBS) containing 0.3 nM BC200 RNA and 1–6 μg eIF4A for 30 min at room temperature. The reaction mixture was subjected to dot-blot analysis, as described previously (Jung et al., 2014 (link)), using two membranes of Hybond ECL membrane (GE Healthcare, USA) and Hybond-XL membrane (GE Healthcare) after which each membrane was washed two times with PBS. The Hybond-XL membrane was hybridized with ³²P-labeled anti-BC200 probe (5′-TTT GAG GGA AGT TAC GCT TAT-3′) and exposed to the BAS-IP imaging plate (Fujifilm, Japan) and analyzed on an FLA-7000 phosphor-image analyzer (Fujifilm). The amount of radiolabeled RNA on each filter was quantified using ImageJ software (NIH, USA).