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

Figure 4.

Figure 4.

AUF1 increases CD83 expression not by altering CD83 mRNA distribution or stability, but by enhancing de novo protein synthesis. (A) COS7 cells were transiently transfected with a CD83 expression construct and were co-transfected with vectors encoding for the indicated AUF1-isoforms. Total and cytoplasmic RNAs were prepared and subjected to PCR analyses (top panels: lanes 1–5: total RNA; lanes 6–10: cytoplasmic RNA; lanes 1 and 6: without AUF1; remaining lanes with indicated AUF1-isoforms; lower panels: qPCR quantification of the CD83 mRNA levels detected in the top panels, normalized to GAPDH levels). (B) HeLa-tTA cells were transfected with a construct encoding inducible CD83 cDNA and were co-transfected with a vector encoding AUF1p40 or a parental vector for control. After a transcriptional pulse, a time course was performed to isolate residual CD83 mRNA and calculate the half-life of CD83 transcripts by quantitative real time PCR. GAPDH-normalized CD83 mRNA levels are depicted. (C) COS7 cells were transfected with a vector encoding for complete CD83 wt cDNA or CD83ΔPRE cDNA and were co-transfected with eukaryotic expression vectors encoding for FLAG-tagged AUF1-isoforms. Twenty-four hours post–transfection, de novo synthesized proteins were labelled with a 35S-translabel pulse (1 h), crude extracts were subjected to CD83 immunoprecipitation followed by SDS–PAGE. Precipitated CD83 protein was quantified using a phosphorimager and the percentage values indicated in the figure (upper part: CD83 wt; lower part: CD83ΔPRE; lane 1/7: negative control; lane 2/8: without AUF1; lanes 3–6 and 9–12: AUF1-isoforms). (D) Co-transfection of CD83 and AUF1p40 expression vectors and metabolic labelling were performed as described in (C). For chase, cell cultures were maintained in label-free medium for the indicated periods. Protein stabilities were quantified by phosphorimaging (lane 1: negative control; lane 2–7: no AUF1 overexpression; lane 8–13: AUF1p40 overexpression; lanes 2 and 8: newly synthesized CD83 protein; residual lanes: indicated time points post labelling). Lower panel: relative CD83 protein levels over time reveal similar half-lives with (black square) or without (grey rhombus) AUF1p40 overexpression.