4.12. Streptavidin Pulldown Assay
The CSFV IRES or PRRSV 3ʹ-UTR fragment was amplified and cloned into the pcDNA3.1(+) vector to generate pcDNA-IRES or pcDNA-PRRSV-3ʹ-UTR. Subsequently, the plasmids were linearized by EcoRV and transcribed in vitro using a RiboMAX™ large scale RNA production system (catalog no. P1300; Promega) according to the manufacturer’s instructions. The resulting RNA was labeled with photobiotin (catalog no. A14216; Baoman, Shanghai, China) using a mercury vapor lamp for 30 min. HEK293T cells grown on 6-well plates were transfected with the indicated plasmids (pMyc-NS5A and pMyc-eEF1A) and lyzed with the lysis buffer. The lysate collected from two wells of the 6-well plate (lyzed with 150 μL of lysis buffer per well) was incubated with the biotinylated IRES RNA (2 μg) for 4 h at 4 °C, and then incubated with streptavidin beads (catalog no. 11205D; Invitrogen) for another 30 min at room temperature. The beads were washed three times with lysis buffer and analyzed by immunoblotting with the anti-Myc MAb (1:1000). For competitive RNA pulldown, the cell lysate was incubated with an increased amount of unlabeled IRES RNA (0, 0.75, 1.5 and 3 μg) for 1 h at 4 °C and then incubated with the biotinylated IRES and streptavidin beads. The experiments were carried out in triplicates.