Figure 5.

siRNA mediated knock-down of AUF1 reduces CD83 expression in a post-transcriptional manner without affecting mRNA stability. (A) COS7 cells were stably transduced with lentiviral vectors encoding siRNA directed against AUF1. Two weeks after transduction, these cells were transiently transfected with CD83 expression vectors. Forty-eight hours after transfection, metabolic labelling of newly synthesized proteins was performed, crude extracts were subjected to immunoprecipitation and western blot analysis, detecting α-tubulin, AUF1 and the respective de novo synthesized CD83 proteins [left panel: lane 1: CD83 and AUFsiRNA#2; lane 2: CD83 and AUFsiRNA#1; lane 3: CD83 and SD control siRNA; lane 4: SD control siRNA without CD83; CD83 de novo expression quantities are indicated]. (B) HeLa-tTA cells were stably transduced with lentiviral vectors encoding control siRNA (shSD) or shRNA directed against AUF1. Two weeks after transduction, the cells were transfected with a construct encoding inducible CD83 cDNA. After a transcriptional pulse, a time course was performed to isolate residual CD83 mRNA and calculate the half-life of CD83 transcripts by real-time PCR. GAPDH-normalized CD83 mRNA levels are plotted against time. (C) Stably transduced COS7 cells with lentiviral vectors encoding shRNA directed against AUF1 or shSD for control were transfected with an expression vector encoding CD83ΔPRE cDNA. Forty-eight hours after transfection, the cells were treated as described in (A), and de novo synthesized CD83ΔPRE protein was quantified (lane 1: without CD83; lane2: shSD control with CD83ΔPRE; lane 3: shAUF1 with CD83ΔPRE).