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Fig. 4. EM of accumulated secreted alkaline phosphatase (SEAP) activity in uninfected and poliovirus-infected cells. (A-E) Huh7 cells were transiently transfected with SEAP-expressing plasmid pGeneGripTM for 16 hours and either (A) mock-infected or (B-E) infected with poliovirus for 4 hours at an MOI of 30 PFU/cell. Cells were fixed and incubated with a solution containing glycerol phosphate and lead citrate, so that alkaline phosphatase activity would lead to the formation of electron-dense lead citrate. Samples were either processed for (A-D) conventional embedding in epoxyresin or (E) cryosectioning. Thawed cryosections were immunogold-labelled with an affinity-purified polyclonal rabbit anti-ERGICp53 antibody followed by a 15-nm gold-conjugated protein-A incubation. Ultrathin sections (
60-80 nm) of all samples were analyzed by EM. Low magnification confirmed that cells shown in B-E were infected, as evidenced by condensed nuclear morphology and the presence of numerous virus-induced vesicular structures (data not shown). Empty arrowheads mark budding structures at ER membranes filled with electron-dense precipitate. Empty arrows identify small (80 nm) vesicles filled with precipitate. Stars mark precipitate-filled tubular-vesicular structures. (E) Solid arrowheads identify 15-nm gold particles; solid arrows identify precipitate-filled tubular-vesicular structures. ER, structures morphologically consistent with endoplasmic reticulum; dmv, double-membraned vesicle.
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