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. 2015 Jun 11;290(31):18904–18913. doi: 10.1074/jbc.M115.658864

FIGURE 5.

FIGURE 5.

Mutating key residues in SID-1 ECD (A173T or P199L) decreases RNA transport efficiency by full-length SID-1. A, a time line for a dsRNA uptake assay in S2 cells. To study dose-dependent RNA transport by SID-1, different concentrations of 500-bp dsRNA (encoding firefly luciferase sequence) were added at 48 h. RNA treatment time was fixed at 24 h. B, quantification of concentration-dependent dsRNA uptake by different SID-1 variants (n = 3; ±S.D. (error bars)). The percentage of relative luciferase reduction is calculated by 1 − L/Lfree. Lfree is the control luciferase level without adding dsRNA. C, expression of different FLAG-tagged SID-1 variants was detected by Western blot using anti-FLAG antibody. Endogenous actin was detected by anti-actin antibody as loading control. D, a modified time line for dsRNA uptake assay in S2 cells. To study time-dependent RNA transport by SID-1, a fixed concentration of dsRNA (1 ng/ml) was added at 48 h. After various dsRNA treatment times, dsRNA was removed by washing with culture medium. E, quantification of time-dependent dsRNA uptake by different SID-1 variants (n = 3; ±S.D.). dsRNA treatment times tested were 0, 0.5, 1, 2, 4, 6, 12, and 24 h. F, kinetic study of dsRNA uptake by SID-1 variants using shorter dsRNA treatment times (0, 1, 2, 5, 10, 20, 30, and 60 min).