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. 2012 Nov 17;41(1):206–219. doi: 10.1093/nar/gks1069

Figure 2.

Figure 2.

All isoforms of AUF1 bind specifically to CD83-PRE RNA. (A) Schematic depiction of AUF1 isoforms. (B) GST-AUF1 isoforms were expressed in E. coli, purified and analysed by Coomassie stained SDS–PAGE. Lane 1: marker; lane 2: GST-AUF1p37; lane 3: GST-AUF1p40; lane 4: GST-AUF1p42; lane 5: GST-AUF1p45. (C) CD83 PRE RNA was incubated with increasing amounts of the indicated AUF1 isoforms (lanes 1, 5, 9 and 13: 800 ng GST; lanes 2–4, 6–8, 10–12 and 14–16: 100–800 ng of recombinant GST-tagged AUF1 protein). Binding of the proteins was evaluated by RNA gel shift experiments. Right panel: quantification of the gel shift experiment by densitometry. The relation of complexed CD83 mRNA to total CD83 mRNA (bound and free RNA) was plotted against the AUF1 protein concentration as a measure for binding efficiency. (D) Five hundred nanograms of GST-AUF1p37 protein (lanes 2–13) was incubated with radiolabelled CD83 PRE RNA plus increasing amounts of non-labelled competitor RNA. Binding of AUF1p37 to radioactive CD83 PRE RNA was measured as aforementioned (lane 1: 500 ng GST; lanes 2, 6, 10: no competitor RNA; lanes 3–5: CD83 PRE RNA; lanes 7–9: HIV1-RRE RNA; lanes 11–13: TNF-α ARE RNA: each 1-, 2- and 3-fold excess). Right panel: radioactive signals from the shifted labelled CD83 PRE RNA were quantified by phosphorimaging and plotted as binding curves. (E) Thousand nanograms of indicated AUF1-proteins were incubated with CD83 wt mRNA (full length) or CD83ΔPRE mRNA. Complex formation was analysed by gel shift experiment. Lanes 1 and 6: GST-control, residual lanes indicated GST-AUF1 fusion proteins and indicated mRNA binding substrates. (F) Thousand nanograms of GST-AUF1p37 protein was incubated with radiolabelled CD83 PRE RNA (lanes 2–8). Increasing amounts of AUF1-specific antibody or non-specific antibody (anti-FLAG, Sigma) (500–1500 ng each) were added to the reactions. RNA gel shift analyses were performed to detect the binding and supershift of the protein:RNA complexes (lane 1: 1000 ng GST; lane 2: no antibody; lanes 3–5: anti-AUF1 antibody; lanes 6–8: anti-FLAG antibody; lane 9: only anti-AUF1 antibody without AUF1). (G) Radiolabelled CD83 PRE RNA (lanes 1 and 2), and single subloop deletions CD83 PRE ΔSubL1 (lanes 3 and 4); CD83 PRE ΔSubL2 (lanes 5 and 6) and CD83 PRE ΔSubL3 (lanes 7 and 8) were incubated with 1000 ng GST or 1000 ng GST-AUF1p37 as indicated (−/+). RNA gel shift analysis was performed to indicate the binding capacity.