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. 2006 May 5;34(8):2355–2363. doi: 10.1093/nar/gkl277

Figure 1.

Figure 1

The TPO wt mRNA is not subjected to NMD. (A) Schematic representation of the TPO minigene used in this study. Boxes and lines represent exons and introns, respectively. The bold arrow (8) marks the position of the translation initiation codon for TPO. Numbers and arrows indicate the position of the seven uORFs of TPO. The minigene contains uORF 7 and the indicated parts of exons 3, 4 and 5. A FLAG-tag for immunoblot detection is present at the 3′ end of the minigene. The positions of the ΔG and ΔATG 7 mutations are indicated. The arrows below the different construct schemes represent the major translation products of the respective mRNAs. (B) Northern blot of cytoplasmic RNA from HeLa cells transfected with the TPO minigene wt (lane 2) and mutants (ΔG, lane 3; ΔATG 7, lane 4; ΔIntron 4, lane 5). Messenger RNA levels were calculated after normalization to the level of the control mRNA (β-globin+300+eIII) to control for variations in transfection efficiencies and gel loading. Signals were quantified as described in Materials and Methods. Percentages represent mean values that were calculated from at least three independent experiments. (C) Immunoblot analysis of TPO protein expression. The TPO protein was detected with an anti-FLAG antibody. The asterisk indicates a nonspecific cross-reactive band. To control for equal transfection efficiency and gel loading, the blot was reprobed with a β-globin-specific antibody that detects the β-globin control protein (β-globin+300+eIII). (D) Northern blot analysis of total cytoplasmic RNA from cells that were transfected with siRNAs directed against hUpf1 to specifically deplete endogenous hUpf1 protein (lanes 4–6, 9, 10). Cells transfected with siRNAs directed against luciferase (lanes 1–3, 7, 8) served as controls. 20 h after siRNA treatment, cells were cotransfected with TPO minigene constructs wt (lanes 1 and 4), ΔIntron 4 (lanes 2 and 5), ΔG (lanes 3 and 6) and β-globin wt (lanes 7 and 9) and NS 39 (lanes 8 and 10) together with the control plasmid. (E) Immunoblot analysis of HeLa cell extracts transfected with hUpf1 siRNA shown in (D). Immunoblotting was performed with a hUpf1-specific antibody (lower panel). The membrane was reprobed with a tubulin-specific antibody (upper panel) to control for equal loading. (F) Northern blot of cytoplasmic RNA from HeLa cells transfected with the TPO minigene wt (lanes 1 and 4) and mutants (ΔIntron 4, lanes 2 and 5; ΔG, lanes 3 and 6) together with the control plasmid. Cycloheximide (CHX, 12 µg/ml) was added to the cell culture medium 5 h prior to harvest. CHX-treated (lanes 1–3) or untreated (lanes 4–6) cells were transfected, washed and harvested simultaneously. Expression of the mRNAs was analyzed as described in Figure 1B.