Northern Blot Assay

PK-7 cells were transfected with the pIRES-RD114-TR or SIN-RD114-TR TV plasmid encoding either RD114-TRWT or RD114-TRco. Forty-eight hours after transfection, total, nuclear, and cytoplasmic RNAs were extracted by Trizol reagent (Life Technologies) following the manufacturer’s instructions and analyzed by northern blot assay. Five micrograms RNA/sample was run on 0.8% agarose-formaldehyde gel, transferred onto a Hybond-N membrane by capillary transfer, and finally probed with 1 × 10⁶ dpm/mL of a ³²P-labeled 550-bp RD114-TRWT or RD114-TRco probe in PerfectHyb Plus hybridization buffer (Sigma-Aldrich). Membranes were extensively washed and then exposed to X-ray films at –80°C or to a Typhoon Phosphorimager 9000 (GE Healthcare) for direct quantification of the radioactive signal. After stripping, membranes were re-hybridized with an internal control probe encompassing the packaging sequence (ψ) to detect full-length mRNAs.