Anti ar pg21

The expression of genes was measured using real-time RT-PCR analyses with Taqman one-step RT-PCR reagents (Thermo Fisher Scientific) and results were normalized to co-amplified GAPDH. The primer and probe sets for the following genes: MCM2, MCM7, FANCI, BLM, TK1, PCAT1, and GAPDH were purchased as inventoried mix from Applied Biosystems at Thermo Fisher. For immunoblotting, cells were lysed with RIPA buffer with protease inhibitors (Thermo Fisher Scientific) and anti-ZBTB7A (Bethyl), anti-AR PG21 (Millipore), anti-Rb, anti-E2F1 (Cell Signaling), anti-V5, anti-HA (Sigma), anti-HDAC1, anti-GAPDH, or anti-β-actin (Abcam) antibodies were used. Immunoblotting results shown are representative of at least 3 independent experiments.

LNCaP cells were grown in phenol-red-free RPMI-1640 medium supplemented with 10% FBS. LNCaP cells were transfected with P3xFlag-BUD31 plasmid using the Superfect reagent (Qiagen, Valencia, CA), as described by the manufacturer. After transfection, the cells were cultured overnight in phenol-red-free RPMI-1640 supplemented with 8% charcoal-dextran–stripped FBS. Cells were treated with or without 10 nM DHT for 2 h before cross-linking for 10 min with a final concentration of 1% formaldehyde in growth media. Cells were washed twice with ice-cold PBS and pelleted. Chromatin immunoprecipitation (ChIP) assays were performed using a Magna ChIP kit (Millipore, Billerica, MA). Antibodies used in these experiments were anti-AR (PG-21; Millipore), anti-BUD31 (H-61; Santa Cruz Biotechnology), anti-Flag M2 (Sigma), and anti-RNA polymerase II (clone CTD4H8; Millipore). Two microliters of DNA extract (out of a total of 50 μl) was used in each PCR. The primer pairs used were as follows: ARE I/II (-459 to -121), 5′-GCC AAG ACA TCT ATT TCA GGA GC-3′ (forward) and 5′-CCC ACA CCC AGA GCT GTG GAA GG-3′ (reverse); and ARE III (-4288 to -3922), 5′-GGG GTT TGT GCC ACT GGT GAG-3′ (forward) and 5′-GGG AGG CAA TTC TCC ATG GTT-3′ (reverse) (Shatkina et al., 2003 (link)).