The effects of RB on function of NF-κB <i>in vitro</i>.

<p>(A) RB dosage-dependently inhibited the nuclear localization of p65 Lysates from cytoplasm and nucleus respectively after treating of PC3 cells with RB for 24 h were used for western blot. GAPDH and H1 respectively served as the loading control. (B) Immunofluorescence analysis of the inhibitory nuclear localization of p65 by RB-treatment for 12 h in PC3 cells. For confocal microscopy, α-tubulin and p-p65 were immunostained, with nuclei stained with DAPI. (Scale bar, 10 μm). (C) Pretreatment of PC3 cells with RB inhibited binding of nuclear extracts to the NF-κB binding site, as detected by electrophoretic mobility shift assay. Lysates from nuclei after treating of PC3 cells with RB for 24 h were used for EMSA. (D), (E) and (F) RB decreased NF-κB activation in PC3 and DU145 cells, and inhibited LPS-induced NF-κB activation in LNCaP cells. The inhibition was more significant when pNF-κB-Luciferase and RELA expression vector co-transfected. Transiently transfected cells were preincubated with RB for 24 h. The luciferase assay was done as described in <i>Materials and Methods</i>. In (D)–(F), results are the mean ± S.E. of three independent experiments, each performed in triplicate. p<0.05 (*), p<0.01 (**), p<0.001 (***) versus RB-untreated control group respectively.