Hrp conjugated anti mouse immunoglobulin secondary antibody

Cells seeded at 5.7 × 10⁴ in 8-well chamber slide (Permanox; Nunc) were mock infected or infected with ZIKV at an MOI of 1. Immunocytochemistry was performed as previously described using 1:200 anti-flavivirus group E antigen antibody (clone 4G2; Millipore) or an isotypic control and 1:500 HRP-conjugated anti-mouse immunoglobulin secondary antibody (Cell Signaling Technology) (68 (link)). Stained cells representing infected cells were counted. The percent infection in live cells was calculated as the number of stained cells over total number of cells counted. Percent infection in live and dead cells was calculated as [(% viability at 24 h − % viability at 48/72 h) × 100 + (% viability at 48/72 h × % of infection in live cells at 48/72 h)]/% viability at 24 h.

De novo protein synthesis was measured using surface sensing of translation (SUnSET) (70 (link)). A549 cells seeded at 5.4 × 10⁵ cells per well of a 6-well plate were mock infected or infected with UV-inactivated or live ZIKV for 24, 48, and 72 h. As a positive control, cells were treated with 20 μg/ml cycloheximide or its ethanol solvent control for 3 h. Puromycin (10 μg/ml) was added to culture medium an hour before harvesting in RIPA buffer. Equal amounts of proteins were separated on TGX stain-free gels (Bio-Rad) by SDS-PAGE and probed against 1:1,000 antipuromycin antibody (clone 12D10; Millipore) followed by 1:1,000 HRP-conjugated anti-mouse immunoglobulin secondary antibody (Cell Signaling Technology). The total intensity of puromycin bands in each lane was quantified using ImageLab 6.0.1 software and normalized against total protein on the TGX stain-free gel. Percentage of protein synthesis was calculated as a ratio of normalized puromycin band intensity in infected or UV-inactivated virus samples to their respective mock-infected controls at the same time point. Percentage of normalized protein synthesis in the cycloheximide-treated sample is expressed as a ratio to that of an ethanol solvent control.