Figure 6. The characterization of suppressive sequences in the pol and env-nef regions.

(A) The effect of each anti-Drosha siRNA (siRNA1 and siRNA2) on Drosha protein level was evaluated by western blot using an anti-Drosha antibody. The reduced amount of Drosha protein in the Jurkat cells was confirmed at 48 h after treatment with anti-Drosha siRNAs compared with the control or mock treatment. After stripping, the membrane was reprobed with an anti-ß actin antibody. (B) Schematic representation of the experimental procedures for RNAi experiments in Jurkat cells. (C) The effects of the anti-Drosha siRNA at the repressed “a ” and “b” sites in the pol region (element 1 in Fig. 5A) and sites “c”, “d” and “e” in the env-nef region (element 15 in Fig. 5A) were assessed in Jurkat cells (black bars). The derepressive effect was confirmed in each region relative to the corresponding vectors with mutations in the repressive sites (mutation 1-2m-2 in the pol region (lattice bars) and 15-3m-2 in the env-nef region (blue bars); see also Fig. 5B–G). The bar patterns correspond to the mutational patterns shown in Fig. 5. The Renilla/firefly luciferase value was assessed, and the data shown are the mean percentages ± S.D. of the mutated vector from six independent experiments. ***P<0.001. (D) The Jurkat cells were transfected with each vector, and RNA-immunoprecipitation (IP) using anti-human Ago2 (hAgo2) antibody was performed. The input and the IP samples were adjusted equal volume and the same volume was loaded and analyzed by western blot. The levels of the immunoprecipitated Renilla luciferase mRNAs compared to Firefly luciferase mRNAs produced from the vectors (Vectors 1 and 15) and the mutated vectors (Vectors 1-2m-2 and 15-3m-2) were analyzed by RT-qPCR. The normalized values of the Renilla/firefly luciferase levels are shown. The each mutated vector was set to 100.