LNCaP cells and DNA from LNCaP cells, the vehicles chip was carried out using PCR subtraction genes Fisher system, with some modifications. Briefly, DNA chip-vehicle-treated LNCaP cells was blunted, ligated to Lenvatinib linkers with BamHI site and amplified by PCR, followed by treatment with BamHI to remove linker. The chip DNA from R1881-treated LNCaP cells was also blunted, ligated to amplified a different set of linkers with BamHI site and treated with DNA from cells with ChIP vehicle for subtractive hybridization. After hybridization, the unhybridized DNA by PCR was amplified. The linkers were removed by digestion of BamHI. This procedure was repeated twice for the specificity of t AR ChIP to improve DNA binding. DNA fragments were cloned into Bluescript vector and sequenced. We have used an analysis of the DNA sequence in more than 100 clones. For the determination of their genomic location by BLAST search, we identified 69-fragments that are less than 50 kb from the transcription start site genes or genes are distributed in RefSeq RefSeq. Sequences for human RefSeq transcripts were obtained from the UCSC Genome browser. Analysis of the enrichment of androgen has been accessed using a matrix LY2109761 method of the weighted position using the matrices from position weighted TRANSFAC. The score threshold was first pattern shown 4 against the background. Among them, we spent 30 primers targeting genomic regions of CHIP putative ARBS for validation by the implementation of the classic chip analysis. We observed ligand-dependent Independent increase in AR binding at least about 12 AR-binding sites. We chose the five best ARBS for further studies. A quantitative RT-PCR Total RNA was prepared using ISOGEN reagent.
First strand cDNA was prepared using the Prime Script RT reagent kit according to claim manufacturer’s protocol. The primer sequences for AR and TACC2 in 4 erg Listed Complementary table. MRNA was quantified by real time PCR using SYBR Green PCR mix and ABI Prism 7000 system on SYBR Green fluorescence. The evaluation of the relative differences in amounts of PCR products between the treatment groups was carried out by the method of Comparative Example cycle threshold, wherein glyceraldehyde 3-phosphate dehydrogenase as contr The house. Quantitative PCR chip accumulation times for IgG-or vehicle-controlled comparison Were quantified by real-time PCR. Differences in the amount of PCR products between the treatment groups were assessed by the method of Comparative Example cycle threshold and as a means of contr glyceraldehyde 3-phosphate dehydrogenase The house. The primer sequences for ARBS 1 can be in ergs Complementary listed in Table 4. The primer sequences for 3 and controls the ARB Positive PSA enhancer described above. Patients and tissue samples from surgical specimens of prostate cancer were obtained from Tokyo University, h Obtain consent from patients for capital. The study was approved by the Ethics Committee of the University t of Tokyo. The Daunorubicin clinical and pathological data from patients in ergs Complementary listed in Table 2. Immunohistochemistry Formalin-fixed tissues were embedded in paraffin and sectioned. A Histofine Kit, which biotin-streptavidin-coated amplification ftigt Was used for immunohistochemical analysis of TACC2. Antigen retrieval was performed by heating the films in an autoclave at 120 ° C for 5 min.