MAVS Localization During EBV Lysis

MAVS cDNA (DNASU#HsCD00296475) was cloned into pDEST-CMV-N-EGFP plasmid (a gift from Robin Ketteler, Addgene plasmid #122842^{110 (link)} by Gateway cloning to produce pDEST-CMV-N-EGFP-MAVS plasmid. To visualize the mitochondria, the mito-BFP plasmid (a gift from Gia Voeltz, Addgene plasmid #49151^{111 (link)} was used. Mito-BFP expresses BFP fused to an N-terminal mitochondrial targeting signal sequence obtained from COX4 amino acids 1–21. AGSiZ cells were plated in glass bottom 35 mm dish (ibidi #81158) at 0.4 million/ml. The next day, the plated cells were replenished with fresh medium at least 1h prior to co-transfection by pDEST-CMV-N-EGFP-MAVS and mito-BFP plasmids using Lipofectamine 2000 (Thermo) according to manufacturer’s instruction. Transfection medium was replaced by fresh medium after 6h and cells were incubated for an addition of 18h before live cell imaging. EBV lytic cycle was induced with 10 μg/ml Doxycycline and active lysosome was stained by 200 nM SiR-lysosome (#CY-SC012, Cytoskeleton, Inc.) during the course of experiment. At 200 mins post-lytic induction, image acquisition was performed with Zeiss LSM 800 instrument at an interval of 10 s for 15 mins and Zeiss Zen Lite (Blue) software was used for image analysis.