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This subline has been previously established in our laboratory by long-term culture in an androgen-deprived medium, thereby gaining characteristic features of androgen ablation-resistant prostate cancer. Downregulation of the AR was achieved with 3 different siRNAs (siAR-1, siAR-2 and siAR-3). For siRNA-mediated knockdown of PKA RI��, we used a target sequence, which corresponds to a previously described antisense oligonucleotide that has already reached clinical evaluation.22, 28, 29 Concentrations of 20 nM of each siRNA were sufficient to significantly reduce protein expression of AR and PKA RI��, respectively, in all 3 cell lines, LNCaP (Fig. 2a), LNCaPabl (Fig. 2b) and VCaP (Fig. 2c). Hence, target knockdown worked in androgen-sensitive as well as in androgen ablation-resistant prostate cancer cells irrelevantly of AR expression levels. The siLUC negative control did not affect AR expression, suggesting the specificity of the siRNAs. http://www.selleckchem.com/products/z-vad-fmk.html Because PKA-I forms a tetramer but the siRNA in this study was only directed against the regulatory subunit of the protein, we further assessed whether depletion of PKA RI�� is sufficient to inhibit PKA signaling. To do so, we determined phosphorylation of VASP (pVASP), a specific downstream substrate molecule of PKA and phosphorylation of cAMP response element-binding protein (pCREB) another main target of activated PKA in LNCaP cells. As summarized in Figure 3, pVASP was significantly diminished by siRNA-mediated http://www.selleckchem.com/products/ly2157299.html knockdown of PKA RI�� by 75% versus control (Fig. 3a, p http://www.selleck.cn/products/XL184.html (Fig. 3b). Moreover, PKA activity was significantly reduced to 53% in comparison with mock control (Fig. 3d, p = 0.001). In contrast to siPKA, the siLUC control had only a minor effect on pVASP expression or pCREB and did not affect PKA activity. Within the same setting, forskolin (5 ��M for 48 hr) was used as a positive control showing induction of PKA activity up to 157% (Fig. 3d, p