Figure 8.

Caspase-2 expression is regulated by DHT. (a) C4-2 and LNCaP were cultured for 48 h in the presence of the indicated doses of DHT. Caspase-2 expression was estimated by Western blot analysis. (b) DHT regulates the expression of endogenous but not ectopically expressed caspase-2. LNCaP-mock and LNCaP-casp-2-mut cells were treated for 24 and 48 h with 10 μm of DHT. Caspase-2 expression was detected by Western blot analysis. (c) PC3-Hygro (mock) and three different PC3-transfected cell lines with wild-type AR were treated with 10 μm DHT and caspase-2 expression was examined by Western blot analysis. (d) The caspase-2 gene contains ARE in intron regions and binds to the AR in vivo. PCR was performed on DNA specifically complexed to the AR protein. Two primer sets were designed to cover the c2-ARE1 and c2-ARE2 that should produce amplicons of 146 and 210 bp in size. PCR was performed in the presence of [α-³²P]-dATP, using primers for c2-ARE1 and c2-ARE2, on DNA extracted after ChIP procedure with antibodies to AR. Samples were separated on 6% PAGE and gels were dried and exposed on X-ray film for 12 h at room temperature. 1 and 5 – positive control with non complexed DNA before immunoprecipitation; 2 and 6 – control immunoprecipitation procedure without antibodies; 3 and 7 – chromatin DNA immunocomplexed with AR antibody; 4 and 8 – chromatin immunoprecipitation with mouse IgG