AR protein levels and activity depending on MID1. (A) AR-dependent CAT reporter gene assay in presence of different androgen (R1881) concentrations. PC-3 cells were co-transfected with plasmids expressing AR and either wild type MID1, mutated MID1 (del1313TGAT) or empty plasmid as control. CAT activity was measured in counts per minute (cpm) by using the reporter gene construct (ARE)2TATA-CAT. Error bars in barplots denote standard deviation (n = 4). (B) Western blot analysis detecting AR and GAPDH proteins after co-transfecting PC-3 cells with AR and either MID1, MID1 1313delTGAT or empty vector as control. Densitometric analysis of respective Western blots from several experiments as indicated. (C) Luciferase reporter assay showing that the MID1-binding sites on AR-mRNA are responsible for the MID1-dependent translational enhancement of AR: PC-3 cells were transfected with either control reporter vector or a construct with the polyCAG-region or the polyGGY-region of the AR-mRNA cloned into the 3′UTR of the luciferase reporter gene. Luciferase activity was measured and the values relative to the control and normalized to the luciferase-mRNA levels are shown (n = 3).