3′UTR cloning and target analysis of miR26b*/RelA and miR562/NF-κB1.

WT and mutant (A) RELA and (B) NF-κB1 3′UTR were subcloned into psiCHECK-2 luciferase vector. The predicted miR binding sites within the respective 3′UTRs is shown. The mutated binding site is represented by asterisks (*). (C,E) 293T cells were transfected with the RELA or NF-κB1 3′UTR plasmid or the mutant RELA or NF-κB1 3′UTR plasmid together with their respective miRs. (D, F) PCR analysis of RELA or NF-κB1 expression after transfection with their respective miRs. (G, H) Western blot analysis of NF-κB protein products p65 and p105/50 after transfection with their respective miRs. A positive control of MCF7-V5 cells overexpressing ANXA1 is shown in lane 3.