3′-UTR of the 1-141 isoform of PTHrP mRNA downregulated luciferase expression in HARA cells. A. Nucleotide sequence of the 3′-UTR of PTHrP 1-141 mRNA. AREs are underlined, core ARE sequences are capitalized. Small region used in transient transfections is indicated in bold face. B. Diagram of luciferase-3′-UTR constructs used in transient transfections. The 3′-UTR of PTHrP 1-141 mRNA in both orientations or the small region of the 3′-UTR containing AREs were subcloned into the XbaI site of pGL3 control vector between the luciferase open reading frame (ORF) and SV-40 3′-UTR sequence. C. Transient transfections of luciferase-3′-UTR reporter constructs into HARA cells. HARA cells were transfected with 2 μg of either pGL3control vector or pGL3control vector containing either the 1-141 isoform 3′-UTR in either direct (pGL3c-UTR) or reverse orientation (pGL3c-rUTR) or the smaller 3′-UTR region (pGL3c-ARE). Luciferase activity was assayed, normalized to protein concentration and plotted. pGL3c-UTR was significantly lower than pGL3c (*, p-value = 0.0002), pGL3c-ARE was significantly lower than pGL3c (**, p-value = 0.001) and pGL3c-ARE was not statistically different from pGL3c-UTR (p-value = 0.40).