Figure 3

MicroRNA regulation of endogenous PBX3 expression. A) LNCaP cells were transfected with 5·10^-8M miR-101, miR-222, Let-7d or control mimic microRNA (Ctr.) as shown in the left panel. The right panel shows transfection of LNCaP cells with 10^-7M Let-7d mimic Hairpin Inhibitor. Total protein was extracted from the cells 48 hours post-transfection. Western analysis was performed with an anti-PBX3 antibody and anti-α-tubulin as loading control. Representative Western blots are shown and the results from the densitometric analysis of three independent experiments are presented in the histograms above (mean ± SD (n = 3)). A t-test (paired two samples of mean) was performed and a two-tailed P value < 0, 05 is indicated with a *. B) Let-7d mimic transfection of C4-2B and RWPE-1 cells followed by detection of endogenous PBX3 levels by Western analysis. Anti-α-tubulin (C4-2B) and anti-PRKAR1A (RWPE-1) antibodies were used as loading controls. Representative Western blots are presented.