Distribution of Us9 and phospho-Us9 across membrane microdomains.

A.) WB analysis following lipid raft flotation from differentiated PC12 cells at 14 hours post-infection with PRV Becker. Samples were collected from a discontinuous 5%–30%–40% Optiprep gradient. DRMs localize to the 5%–30% interface, while solubilized membrane proteins remain at the 30%–40% interface. Each 1 mL fraction from this gradient was run and probed with polyclonal anti-Us9 antibody to detect total Us9 protein content and phospho-specific monoclonal antibody to detect only phosphorylated Us9. B.) Quantitation of total Us9 and phospho-Us9 in insoluble raft membrane fraction and soluble fraction by WB for PC12 cells 14 hours post-infection with PRV Becker; values are reported as arbitrary chemiluminescence units from WB. Curves are representative of two independent lipid raft flotation experiments and show detection of each sample across a 2-fold dilution series. Dilution series covers detection of the respective Us9 protein from point of saturation, through the linear range of detection, to undetectable levels.