Deletion of individual CD83 PRE substructures impairs binding to HuR.

<p><b>A.</b> GST alone (negative control, 2.2 µM, lane 1, 6, 11, 16) or increasing amounts of recombinant GST-HuR (0.19 µM, 0.38 µM, 0.57 µM, 0.95 µM) were incubated with radiolabelled CD83wt PRE RNA or several combinations of uridine-rich element (URE) mutants as indicated at the top of the panels. Formation of protein-RNA interaction (indicated by arrowhead) was subsequently detected by gel retardation analysis. <b>B.</b> GST alone (negative control; 2.2 µM, lane 1, 5, 9, 13) or increasing amounts of recombinant GST-HuR (0.237 µM, 0.475 µM, 0.95 µM; lanes 2–4, 6–8, 10–12, 14–16, respectively) were incubated together with radiolabelled full-length CD83wt PRE RNA or various PRE subloop (SubL) deletions (depicted at the top of the panels). Complex formation (indicated by arrowhead) was subsequently visualized by gel retardation assay as before. <b>C.</b> COS7 cells were transfected with expression vectors encoding for human CD83 cDNA flanked by the entire homologous 5′- and 3′-UTR (lane 1 and 2) or derivatives thereof (lane 3: ΔSubL1; lane 4: ΔSubL2; lane 5: ΔSubL3). Cellular lysates were subjected to immuno-PCR using anti-HuR antiserum (lane 2–5) or rabbit IgG for negative control (lane 1). CD83-specific RNA was detected by PCR followed by gel electrophoresis. A mock reaction without template served as additional negative control (nc; lane 6). This experiment has been reproduced at least three times with the same results.