Progeny Varicella-Zoster Virus Capsids Exit the Nucleus but Never Undergo Secondary Envelopment during Autophagic Flux Inhibition by Bafilomycin A1

Girsch, James H.; Walters, Katherine; Jackson, Wallen; Grose, Charles

Gradient sedimentation and Western blotting. The protocol for sedimentation of VZV sonicates in potassium tartrate-glycerol gradients has been described previously; the virion bands from the gradients were pelleted, and the supernatant was discarded (52) . The pellets were solubilized in SDS-PAGE sample buffer (0.125 M Tris, pH 6.8, 2% SDS, 10% beta-mercaptoethanol, 20% glycerol, and bromophenol blue). The solubilized, purified virus was separated on 4 to 20% gradient SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with phosphate-buffered saline (PBS) with 0.05% Tween 20 containing 5% nonfat dry milk for 30 min at room temperature and then incubated in primary antibodies diluted 1:2,000 in PBS-0.05% Tween 20-1% milk for 1 h at room temperature. The membranes were washed 5 times for 5 min each time at room temperature in PBS-0.05% Tween 20, incubated in goat anti-mouse or goat anti-rabbit horseradish peroxidase (HRP)conjugated secondary antibodies, diluted 1:25,000 in PBS-0.05% Tween 20-1% milk for 1 h at room temperature, and washed 5 times for 5 min each time at room temperature in PBS-0.05% Tween 20. The labeled proteins were detected using a SuperSignal West Pico Plus chemiluminescent substrate kit from Thermo Scientific.