Actin Assay

The amount of F-actin versus G-actin was measured using the G-actin/F-actin In Vivo Assay kit (Cytoskeleton, Denver, CO), per manufacturer’s protocol as described earlier[37]. Briefly, treated HSV samples were homogenized in 0.25 ml of lysis buffer (50 mM PIPES pH 6.9, 50 mM NaCl, 5 mM MgCl₂ 5 mM EGTA, 5% (v/v) Glycerol, 0.1% Nonidet P40, 0.1% Triton X-100, 0.1% Tween 20, 0.1% 2-mercapto-ethanol, 0.001% Antifoam C, 4 μM Tosyl arginine methyl ester, 15 μM Leupeptin, 10 μM Pepstatin A, 10 mM Benzamidine, 1 mM ATP warmed to 37°C) for 1 min with a mortar and pestle that fit into the 1.5 ml microfuge tube. The lysate (100μL) was centrifuged at 2000 rpm for 5 min at 37°C to pellet unbroken cells. The supernatants were centrifuged at 100,000 g for 1 hr at 37°C. Supernatants (contains the G-actin) were transferred to pre-cooled tubes and placed on ice. The pellets (contain F-actin) were resuspended in 100μL of ice-cold 10 μM cytochalasin D in deionized water, and F- actin was depolymerized by incubating for 1 hr on ice with mixing every 15 min. Equal volume of supernatants and pellets along with actin standards (50-100ng) were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membrane in 1X TG buffer at 100 volts for 1 hr. The membrane was probed with anti actin antibody(1:1000 dilution cytoskeleton) and the amount of actin in each fraction was quantified comparing to actin standards loaded on the same gel.