Western Blot Analysis of Viral Proteins

Immunoblot analysis was carried out to evaluate the accumulation of viral proteins as previously reported [26 (link)]. Briefly, total cells lysates were prepared from cells by SDS sample buffer 1X (62.5 mM Tris-HCl (Tris(hydroxymethyl) aminomethane hydrochloride) pH 6.8; 50 mM DTT (dithiothreitol); 10% glycerol; 2% SDS (sodium dodecyl sulfate); 0.01% Bromophenol Blue; EDTA-free Protease Inhibitor Cocktail 1X (Roche)), and after they are boiled for 5 min. An equal amount of protein extract was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose membranes, and blocked at 4 °C overnight in 5% non-fat dry milk-TBS. GAPDH (sc-32233), UL42 (sc-53333), ICP0 (sc-56985) (Santa Cruz, CA, USA), and US11 (provided by Professor Bernard Roizman) were detected by secondary HRP conjugated goat anti-mouse IgG (Merk, Millipore). Specific bands were visualized using Immobilon Classico Western HRP substrate (Merk, Millipore). Quantitative densitometry analysis of immunoblot band intensities was performed using ImageJ software. The intensity of the target protein was divided by the intensity of the GAPDH and graphically represented by GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).