Protein-Protein Interaction Profiling

Immunoprecipitation was carried out to assess protein–protein interaction^{50 (link)}. Briefly, HEK293T or THP-1 cells were infected with HSV-1 or VSV (MOI = 0.5) for 16 h. Cells were harvested and lysed with NP40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with a protease inhibitor cocktail (Roche). Centrifuged cell lysates were precleared with Sepharose 4B beads and incubated with 10 μl of FLAG-Agarose (Sigma-Aldrich) or 0.5 μg of the indicated antibody plus 10 μl of protein G-Sepharose (GE Healthcare) at 4 °C for 4 h. Agarose beads were washed three times with lysis buffer, and precipitated proteins were released by boiling with 1× sodium dodecyl sulfate (SDS) sample buffer at 95 °C for 5 min. Precipitated proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed by immunoblotting.