3. Total RNA Extraction, Conventional RT-PCR, and Real-Time RT-PCR selleck bio Total RNA was extracted using the TRIzol method (Invitrogen, Carlsbad, CA). Cells (5.0 �� 105) were mixed in a test tube with 1mL TRIzol solution. Prepared RNA was denaturated at 65��C for 15min in a volume of 30��L and cooled on ice for at least 1min. 2.0��g of denatured RNA were then annealed by addition of reaction mixture to a total volume of 20��L (4.0��L of 5 �� RT buffer, 10pmol of primers, 2.0��L of 25mM MgCl2, 2.0��L of 10mM dNTPs, and 0.2��L of 1M DTT in nuclease-free water) and incubated at 42��C for 70min. The reaction was terminated at 95��C for 5min, chilled on ice for 5min, and collected by brief centrifugation. To remove RNA, 1��L of RNase H were added to each tube followed by incubation at 37��C for 20min.

1��L of cDNA were used for each PCR reaction.Amplifications of cDNAs by PCR using specific primer pairs for AR were performed in 20��L reaction volumes containing 10mM Tris-HCl, pH 8.3, 50mM KCl, 1.5mM MgCl2, 0.001% gelatin, 0.2��M each dNTP, 0.2��M of each primer, 1 unit of Taq DNA polymerase (Invitrogen, CA), and 1.0��L cDNA as template.Real-time PCR was performed with an SLAN real-time PCR detection system (LG Life science, Korea) and SYBR Green reagents (Invitrogen, Carlsbad, CA). Specific primers for human GAPDH, AR, HSP27, CLU, GRP78, and c-FLIP were designed to work in the same cycling conditions (50��C for 2min to permit uracil N-glycosylase cleavage, 95��C for 10min, followed by 40 cycles of 95��C for 15s, and 60��C for 1min). We used 1.

0��L of the reverse transcriptase product for PCR in a final volume of 25��L. 2.4. Western BlotPreparation of total cell lysate and the procedures for Western blot analyses were performed essentially as described previously [16]. The antibodies against GRP78, c-FLIP, and AR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody for HSP27 purchased from Millipore (Millipore, MA). The quantity of the applied protein was normalized with anti-actin polyclonal antibody (Sigma Aldrich Korea, Seoul, Korea).Samples with equal amounts of protein (20��g) from lysates of cultured LNCaP cells were subjected to SDS-PAGE and then transferred to a PVDF filter. The filters were blocked in TBS containing 5% nonfat milk powder at 4��C overnight and then incubated for 1h with a diluted each primary antibodies (Actin: 1:10,000; AR, HSP27, CLU, GRP78:1:1,000; c-FLIP: 1:2,000; Santa Cruz, CA).

2.5. Immunocytochemical Analysis and TUNEL StainingCells on coverslips were rinsed 1 �� phosphate-buffered saline (PBS) and then fixed with ice-cold methanol for 15min. Anacetrapib Samples were further permeabilized with PBS containing 0.025% Triton-X detergent (1 �� PBS-TX) for 10min and blocked with 3% BSA in 1 �� PBS for 30min. Cells were reacted with primary antibodies (AR, HSP27, CLU, GRP78:1:100; c-FLIP: 1:50; Santa Cruz, CA) for 1 hour at room temperature.