Electrophoretic mobility shift assay (EMSA) and RNA pull down assay

RNA of EV71-5′UTR was transcribed with a MEGAscriptTM T7 kit (Ambion, Austin, TX, USA), and RNA purified with a MEGA clear kit (Ambion) was annealed with the DNA probe (5′-GTTTAGCTGTGTTAAGGGTCAAG-3′) labeled biotin by a Biotin 3′ End DNA Labeling Kit (Thermo). For EMSA assay, A3G and its mutants with a HA tag were purified by co-IP with anti-HA agarose beads from 5 is × 10⁶ HEK293T cells transfected with A3G or mutant expression plasmid for 48 h, and then incubated with 4 ug biotinylated RNA for the detection of the interaction between EV71-5′UTR and A3G or its mutants with a LightShift^® Chemiluminescent EMSA Kit (Thermo) according to manufacturer's instructions. RNA pull down assay was performed as reported previously (10,43). To be specific, 4 ug biotinylated RNA were heated to 90°C for 2 min to disrupt the secondary structure, and placed on ice for 2 min to form the proper secondary structure for EMSA and pull down assays. The 5 × 10⁶ cells were lysed in lysis buffer described in Co-IP assay supplemented with 20 U Protector RNase inhibitor (Roche), and centrifuged to get the clear cell lysate. Then the lysate were precleared with 50ul Streptavidin-Sepharose Beads (BioVision, Milpitas, CA, USA) by rotating at 4°C for 30 min. Then folded RNA were added to the precleared cell lysate and rotated at room temperature for 1 h. 100 ul beads were added to the reaction and rotated at 4°C for 1 h followed by five washes using the wash buffer described in Co-IP assay supplemented with 20 U Protector RNase inhibitor (Roche). The proteins on beads were detected by western blotting.