FIG 9.

25HC delays reovirus outer capsid cleavage. (A) HeLa cells were pretreated with the vehicle control or 10 μM 25HC for 12 h prior to infection. Cells were then adsorbed with reovirus T1L at 4°C for 1 h. The inoculum was removed, and cells were washed two times with PBS to remove unbound virus and incubated with prewarmed medium at 37°C. At the indicated times after adsorption, the cells were scraped into PBS, pelleted at 3,000 × g, and resuspended in radioimmunoprecipitation buffer. Lysates were clarified by centrifugation at 13,000 × g, resolved in 4 to 12% polyacrylamide gels, and transferred to PVDF membranes. The membranes were probed with a rabbit polyclonal anti-T1L antiserum and an anti-rabbit horseradish peroxidase-conjugated secondary antibody and visualized using chemiluminescence. Data are representative of five independent experiments. (B) HeLa cells were pretreated with the vehicle control or 10 μM 25HC for 12 h prior to determination of cathepsin L (CatL) activity in cell lysates. Error bars represent 95% CI of the results of biological replicates.