Figure 4

The enhanced protein expression caused by tethering Stau2 to the 3'-UTR of an mRNA is mediated by Upf1. 293F cells were transfected with an siRNA for Upf1 (siUpf1-1 or -2) or RHA (siRHA) or with a negative control siRNA (siNC). At 24 h after transfection, an expression plasmid for either MS2 or MS2-Stau2 was co-transfected with plasmids for Rluc-8xMS2bs and Fluc. At 48 h after plasmid transfection, the cells were harvested and subjected to Western blotting (A-C) or the luciferase assay (D-F). (A-C) Western blotting was performed using anti-HA, anti-Upf1, anti-RHA and anti-GAPDH antibodies. (D-F) The expression of Rluc relative to that of Fluc was calculated for each transfection, and the normalized level of the MS2 transfection was defined as 1. Values are expressed as the means ± SEM of four independent experiments.