Pre-miR-886 is physically associated with PKR and suppresses it from activation. (A) Western blotting of PKR (top panel) in the 0.6 M KCl fraction eluted from the indicated biotinylated RNA–streptavidin bead complex (see Materials and Methods). Silver staining of the gel is shown for equal loading (bottom panel). (B) The FLAG–PKR expressing plasmid was transfected to 293T:886 (a 293T derivative cell line stably expressing pre-miR-886 from integrated pLPCX–pre-886). PKR and associated RNA was immunoprecipitated, according to the protocol of Dicer processing assay (see Materials and Methods) with modifications. Coprecipitated RNA with PKR was eluted by 200 mM KCl, purified by Trizol reagent, and subjected to RT-PCR measurement of pre-miR-886, vtRNA1-1, and β-actin mRNA. (C) Western blotting detection of indicated proteins at 24 h after transfection of indicated anti-oligos. All other descriptions are the same as in Figure 5B. (D) Luciferase assays measuring NF-κB activity at indicated hours (x-axis) after co-transfecting reporter plasmids with indicated anti-oligos (left panel) or plasmids (right panel). pNF-κB-Luc plasmid expresses firefly (Photinus pyralis) luciferase under the control of a NF-κB responsive promoter. As a control, we used pcDNA3.1-Zeo(+)-Pp that expresses firefly luciferase under CMV promoter. Each reporter plasmid was co-transfected with pRL-SV40 (Promega) expressing Renilla luciferase for normalization. Each value of the firefly luciferase (Pp) was first normalized to the Renilla luciferase value (Rr), and then the Pp/Rr value of the NF-κB reporter plasmid was again normalized to that of the CMV control (from pcDNA3.1-Zeo(+)-Pp), yielding the relative luciferase activity of the sensor plasmid (y-axis). pBluescript is a negative control; pLPCX–pre-886 expresses pre-miR-886; pIkB-SR expresses constitutively active IκB-α thus was used as a positive control for NF-κB suppression. (E) RT-PCR measurement of NF-κB target genes after transfecting indicated anti-oligos to HCT116 and CRL2741. All other descriptions are the same as in Figure 5B. (F) Luciferase assays at 24 h after transfecting indicated Poly(I:C) and reporter plasmids into 293T:886 (described in panel B) and 293T:vector (a 293T derivative line harboring pLPCX vector, thus lacking pre-miR-886 expression). All other descriptions are the same as in panel D.
