*2.7. Co-Immunoprecipitation and Immunoblotting*

U251 cells (1 × 106), seeded the day before into 10 cm dishes, were infected with PRVABC59 strain of ZIKV (MOI = 1). At 48 h post-infection, cells were washed three times with PBS before lysing with NP-40 lysis buffer (150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol) containing CompleteTM protease inhibitors (Roche, Mannheim, Germany) on ice for 30 min. Lysates were clarified at 16,000 g for 20 min in a microcentrifuge at 4 ◦C. Small aliquots of the clarified lysates were kept for loading controls. The remaining lysates were treated with 20 μg/mL RNase A (Roche; Mannheim, Germany) for 1 h on ice, precleared with protein G-Sepharose beads (Sigma Aldrich; St. Louis, MO, USA) for 1 h at 4 ◦C before sequential incubation with antibodies overnight and then protein G-Sepharose beads for 2 h at 4 ◦C. Rabbit IgG was used in parallel as a negative control. Immunoprecipitates were washed three times with NP-40 lysis buffer before the bound proteins were eluted by boiling in SDS Sample buffer. Proteins were separated by SDS-PAGE and transferred to (PVDF) membranes for immunoblotting.
