4.7. SUnSET-Puromycin Assay

A549 and U251 cells were seeded in 12-well plates and cultured for 48 h. When 80% confluency was reached, cells were inoculated with the Mexico ZIKV strain at 400 TCID 50/mL (or an identical volume of the mock culture). Alternatively, 10 μg/mL or 5 μg/mL of the translation inhibitor cycloheximide (CHX; stock solution 100 mg/mL; Sigma–Aldrich) was added to A549 cells and U251 cells, respectively, or 10 μM eIF4G1 inhibitor (4EGI-1; stock solution 10 mM; Biotechne) [44] was added. At 48 h p.i., cells were washed twice with PBS and incubated in unsupplemented DMEM for 2 h at 37 °C. The supernatant was discarded, fresh DMEM medium supplemented with 3% FBS, and 1 μM puromycin (stock solution; Merck, Rahway, NJ, USA) was added, and cells were further incubated for 30 min at 37 °C. Subsequently, cells were collected for Western blotting analysis.