Cells were scraped and incubated with 100 μl of radioimmunoprecipitation assay buffer with protease phosphatase inhibitor [Halt Protease and Phosphatase Inhibitor Cocktail (100×); Thermo Fisher Scientific] for a period of 30 min on ice. Whole-cell protein extracts (supernatant) were collected by centrifuging the mixture at 13,500 rpm on a benchtop refrigerated (4°C) centrifuge for 15 min. Protein samples were quantified (Thermo Fisher Scientific Bicinchoninic Acid Kit), normalized, and then denatured in NuPAGE LDS Sample Buffer (Invitrogen, NP00007) and β-mercaptoethanol by heating them to 80°C for 10 min. The denatured protein samples were allowed to cool, and equal amounts of protein were added to 4 to 12% SDS–polyacrylamide gel electrophoresis loading gels and run at a constant speed at 70 V for 3 hours. The protein from the gel was then transferred to a nitrocellulose membrane using an iBlot 2 dry transfer instrument (Thermo Fisher Scientific, USA). Nitrocellulose membrane was blocked in 5% nonfat milk in tris-buffered saline and 0.1% Tween 20 (TBST) for 1 hour at room temperature. After the blocking step, membranes were incubated with anti–HSV-1 and anti–HSV-2 gB mouse monoclonal antibody (Abcam, 6506) or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Proteintech, 10494-1-AP) antibody at dilutions of 1:1000 overnight at 4°C. The following day, the blots were washed multiple times with TBST before the addition of horseradish peroxidase–conjugated secondary immunoglobulin G antibody at dilutions of 1:10,000 at room temperature. Protein bands were visualized on an ImageQuant LAS 4000 imager (GE Healthcare Life Sciences) by the addition of SuperSignal West Pico maximum sensitivity substrate (Pierce, 34080). The density of the bands was quantified using ImageQuant TL image analysis software (version 7). GAPDH was measured as a loading control.

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