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

Figure 3.

Figure 3.

Overexpression of all AUF1 isoforms enhances exogenous CD83 expression in a PRE-dependent manner. (A) COS7 cells were transiently transfected with reporter constructs encoding firefly luciferase or a firefly luciferase-PRE fusion together with indicated AUF1-isoforms. For internal control purposes, a vector encoding chloramphenicol transferase (CAT) driven by a CMV-IE promoter was co-transfected. Forty-eight hours post–transfection, crude extracts were prepared, and CAT as well as luciferase activities were analysed. Relative CAT-graded luciferase activities are depicted. (B) COS7 cells were transfected with a vector encoding complete CD83 wt cDNA or CD83ΔPRE cDNA and were co-transfected with eukaryotic expression vectors encoding FLAG-tagged AUF1-isoforms. Forty-eight hours post–transfection, crude extracts were subjected to western blot analyses detecting α-tubulin, AUF1 and CD83 (upper part: CD83 wt; lower part: CD83ΔPRE; lane 1/6: without AUF1; lane 2/7: AUF1p37; lane 3/8: AUF1-p40; lane 4/9: AUF1p42; lane 5/10: AUF1p45). (C) COS7 cells were co-transfected with a vector encoding the indicated AUF1-isoforms, a vector encoding full length CD83 cDNA, as well as with increasing amounts of a vector encoding CD83 PRE RNA (1-, 2- and 3-fold excess). As a control, HIV-1-RRE RNA was transfected instead of CD83 PRE RNA. Forty-eight hours after transfection, crude extracts were analysed by western blot analyses, detecting α-tubulin and CD83 (in each panel: lane 1: without AUF1; lane 2: indicated AUF1 isoform; lanes 3–5: isoform of AUF1 and increasing amounts of competitor RNA).