. A. Western blot (WB) analysis of RGS16 in parental and ORF2-expressing PK15 cells in the absence (empty vector) or presence of let-7e overexpression. α-Tubulin served as an internal control. Mature let-7e levels were also measured by splinted ligation assay. The 5.8S rRNA served as a loading control. Note that let-7e is down-regulated in ORF2-expressing cells (lane 3) relative to parental cells (lane 1). Values below the images indicate quantified signal intensities, and the level of RGS16 protein or let-7e in parental cells transfected with the empty vector was set to 1. B. Semi-quantitative RT-PCR analysis of RGS16 and GAPDH mRNA levels in parental and ORF2-expressing cells in the absence (empty vector) or presence of let-7e overexpression. No significant difference in RGS16 mRNA levels was detected between samples. GAPDH was used as an internal control. C. Relative luciferase activity from the Renilla luciferase-RGS16 3′ UTR reporter in parental and ORF2-expressing PK15 cells (top), and in parental PK15 cells in the absence (empty vector) or presence of let-7e overexpression (middle). The level of mature let-7e in each sample was also determined by splinted ligation assay (bottom). The 5.8S rRNA was used as a loading control. Renilla-luciferase activity was normalized to firefly-luciferase activity, and the normalized activity of Renilla luciferase in parental cells (top) or the same cells transfected with the empty vector (middle) was set to 1. All error bars indicate the mean ± SEM from three independent experiments. **P < 0.01 by the Student’s t-test. Abbreviations: RL, Renilla luciferase; FL, firefly luciferase.