Figure 3.

Staufen1 does not enhance alternative splicing of the dl HIV-1 IRES RNA nor increases the cryptic promoter activity of the dl HIV-1 DNA. (A, B) The dl HIV-1 IRES (150 ng) was cotransfected with a control scRNA (100 nM) or with siRLuc (100 nM), in the presence, or the absence (−), of the Stau155-HA3 (325 ng) plasmid. (A) Schematic representation of the dl reporter targeted by the siRNA RLuc (siRLuc) targeting the Renilla luciferase ORF (upper panel). Total protein extracts were prepared 48 hrs post-transfection. The expression of Stau155-HA3 was determined by western blot, using the GAPDHprotein as a loading control (lower panel). (B) RLuc and FLuc activities were measured and expressed relative to the values obtained with scRNA, set to 100% (RLA). Values shown are the mean (±SEM) for six independent experiments, each performed in duplicate. Statistical analysis was performed by an ordinary two-way ANOVA test (*P < 0.01; ns, not significant). (C, D) HEK 293T cells were transfected with either the dl HIV-1 IRES (150 ng) or a promoterless ΔSV40-dl HIV-1 IRES (150 ng) vector in the presence, or the absence (−), of the Stau155-HA3 (325 ng) plasmid. 24 hrs post-transfection total protein extracts were prepared. (C) Schematic representation of the dl HIV-1 IRES and ΔSV40-dl HIV-1 IRES plasmids (upper panel). The expression of Stau155-HA3 was determined by western blot, using the GAPDH protein as a loading control (lower panel). (D) RLuc and FLuc activities were measured, and results are expressed as RLA relative to the activities obtained from the dl HIV-1 IRES vector when in the absence of the Stau155-HA3, set to 100%. Values shown in are the mean (±SEM) for three independent experiments, each performed in duplicate. Statistical analysis was performed by an ordinary two-way ANOVA test (**** P < 0.0001; ns, not significant).