Tnt r quick coupled transcription translation systems

In vitro translation was carried out with TNT(R) Quick Coupled Transcription/Translation Systems (Promega) according to the instructions of the manufacturer. In brief, 100 ng pET32a-HA-E6SDm or 200 ng pcDNA3.1(+)-E1SDm-3XFlag plasmid was translated in the absence or presence of 1 μM recombinant hnRNP D protein (EUPROTEIN) or 1 μM BSA. The 25 μl reactions were incubated for 90 min at 30°C. The translation reactions were analyzed by western blotting as described above. The Luciferase control RNA was also translated in the absence or presence of 1 μM hnRNP D or 1 μM BSA. Luciferase activity in the translation reactions were monitored according to the instructions of the manufacturer using Tristar LB941 Luminometer. Recombinant hnRNP D and BSA were separated on SDS-PAGE followed by staining with Colloidal Blue Staining Kit (Invitrogen).

The reaction was performed using the TNT^R Quick Coupled Transcription/Translation Systems (Promega, Madison, WI, USA) according to the manufacturer’s instructions using 1 μg of expression construct without or with increasing doses of the corresponding MO (10 ng, 100 ng and 500 ng). The reaction was resolved on a 10% NuPAGE Bis-Tris gel, transferred onto a PVDF membrane using the iBlot system (Invitrogen) and the products revealed using a colorimetric reaction.