Figure 6.

Knock-down of AUF1 attenuates endogenous CD83 expression in a mononuclear cell line at the post-transcriptional level. MonoMac6 cells were stably transduced with lentiviral vectors encoding control siRNA (siSD) or AUF1-siRNA#2. Two weeks later, the cells were activated with ionomycin and PMA. Within several hours, the cells were harvested and (A) endogenous CD83 surface expression was quantified by FACS analysis (left control; right siAUF1 knock-down). (B) CD83 de novo synthesis between 1.5 and 2.5 h post-activation was measured by a transcriptional pulse experiment. The respective crude extracts were subjected to immunoprecipitation, followed by SDS–PAGE and quantification by phosphorimaging. In parallel, the crude extracts were evaluated by western blot analysis of α-tubulin and AUF1 expression (lanes 1 and 2: SD siRNA controls; lanes 3 and 4: siAUF1; lane 1 and 3: no activation; lane 2 and 4: activation). (C) CD83 mRNA transcription rates were determined by isolating and reverse transcribing total RNA from cells during the time course described in (A) and (B). The respective cDNA was quantified by real time PCR, including GAPDH transcripts for evaluation. GAPDH-normalized CD83 transcript signals are plotted against time.