Rabbit anti srd5a1

Cells were starved with phenol red-free and serum free-medium for at least 48 h before treatment with the indicated drugs and/or androgens. RNA extraction and cDNA synthesis were performed with the GenElute Mammalian Total RNA miniprep kit (Sigma-Aldrich) and iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) respectively. Quantitative PCR (qPCR) analysis was conducted in triplicate in an ABI 7500 Real-Time PCR machine (Applied Biosystems) using iTaq Fast SYBR Green Supermix with ROX (Bio-Rad) and primers for TMPRSS2, PSA and RPLPO, as described previously ^{4 (link)}. SYBR Premix Ex Taq II (Takara) was used for SRD5A1, SRD5A2 and AR v7 detection. Primers used for AR detection are 5′-TCTTGTCGTCTTCGGAAATGT-3′ and 5′-AAGCCTCTCCTTCCTCCTGTA-3′ ^{25 (link)}. Primers used for AR v7 detection are 5′-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3′ and 5′-TTTGAATGAGGCAAGTCAGCCTTTCT-3′^{26 (link)}. Accurate quantitation of each mRNA was achieved by normalizing the sample values to RPLPO and to vehicle-treated cells. 50 μg cell lysate was used for immunoblot with rabbit anti-SRD5A1 (Abnova) and mouse anti–β-actin (Sigma-Aldrich) antibodies.