Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.; van Kuppeveld, F. J. M.

Mammalian two-hybrid assay. pACT, pBIND, and pG5Luc (Luc stands for luciferase) vectors were from Promega. pBIND-GBF1 encoding the N terminus of GBF1, pACT-3A, and pBIND-3A were described previously (38, 39) . pACT-ACBD3 was a kind gift from J. Sasaki (Fujita Health University School of Medicine, Aichi, Japan) (45) . pACT-3A(S11aa2) and pBIND-3A(S11aa2) were cloned with a forward primer containing the GFP(S11) tag using standard DNA cloning techniques. Subconfluent layers of COS-1 cells seeded in 24-well plates were transfected with 350 ng of pACT, pBIND, and pG5Luc plasmids using Fugene6 (Promega) according to the manufacturer's protocol. At 48 h p.t., cells were lysed, and Renilla and firefly luciferase levels were measured using the Dual-Luciferase assay kit (Promega) following the manufacturer's protocol. Values were converted to firefly/Renilla signal ratios to correct for transfection efficiencies.