SDS-PAGE and Western Blot Analysis

SDS PAGE and Western blot analysis were performed using standard methods (see for recent comprehensive protocol [96 ]). RSC were infected with mutant and wild type 17Syn+ and at the times indicated (see S1 Fig) infected RSC were lysed and boiled in Laemmli cocktail, loaded onto a 10% polyacrylamide gels and separated by electrophoresis [97 (link)]. Following separation, proteins were transferred to nitrocellulose [98 (link)]. The uniformity of transfer was evaluated by Pounceau S staining of the membrane. Western blot was performed using standard procedures, including blocking of non-specific binding of antibodies in 5% nonfat milk (1 hr), incubation in 2% PBS-BSA solution containing primary antibody (1 hr), followed by rinsing (PBS, 3x15 min), incubation in a solution containing HRP conjugated anti-rabbit antibody (1 hr) (Vector labs), and rinsing (PBS, 3x15 min). The primary antibodies include a rabbit Pan HSV antibody 1:5,000 (Accurate), an affinity purified rabbit anti-VP16 peptide antibody 1:1000 [11 (link)], and HSV-1 anti-ICP0 affinity purified mouse monoclonal (Santta Cruz: 110600) diluted 1:1000 [54 (link),78 (link)]. The peroxidase substrate, VIP (Vector) was utilized according to manufacturer’s protocol. Blots were scanned and analyzed using Image J software.