FIGURE 9.

Steroid receptor synergism with MAGE-11, TIF2, and p300. A, full-length human AR amino acid residues 1–919 and hAR-(1–660), -(1–503), -(507–919), and Δ120–472 deletion mutants contain the NH₂-terminal ²³FXXLFXXVXXV³³ motif, AF1, DNA binding domain (DBD), and AF2 in the ligand binding domain (LBD) as indicated. Human AR-(1–660) corresponds to mAR-(1–640), hPR-B-(1–688), and hGR-(1–550). Human AR-(507–919) corresponds to mAR-(487–899), rGR-(399–795) and hPR-B-(486–933). Human ARΔ120–472 corresponds to mARΔ101–452. B, pCMV-hAR-(507–919) WT or K720A mutant, corresponding pCMV-mAR-(487–899) and K700A mutant, pCMV-rGR-(399–795), and pcDNA-myc-PR-B-(486–933) (0.1 μg) were transfected with 3 μg of MMTV-Luc and 0.5 μg of pSG5 empty vector (−), pSG5-MAGE (M), and/or pSG5-TIF2 (T) in CV1 cells. Cells were incubated with and without 10 nm DHT for AR, 10 nm dexamethasone for GR, and 10 nm progesterone for PR. Inset, pCMV5 empty vector (−), pCMV-hAR-(507–919) and K720A mutant, and corresponding pCMV-mAR-(487–899) and K700A mutant (5 μg) were expressed in COS cells. The transblot of cell extracts (50 μg of protein/lane) was probed using AR52 antibody. C, top panel, endogenous protein extracts (100 μg/lane) from HeLa (HL; lane 2) and CV1 (CV; lane 3) cells were compared with extracts of COS cells expressing pSG5-HA-p300 and pSG5-TIF2 as control (10 μg of protein; lane 1). The blot was probed using p300, TIF2, and β-actin antibodies. Bottom panel, pSG5 empty vector (lane 1) and pSG5-HA-p300 (lanes 2–9) (2 μg) were expressed in COS cells with and without 10 nm nonspecific siRNA-3 (NS3), -5 (NS5), or p300-siRNA-1–4 using Lipofectamine 2000. Cell extracts (15 μg of protein/lane) on the transblot were probed overnight at 4 °C using p300 and β-actin antibodies. D, CV1 cells were transfected with 3 μg of PSA-Enh-Luc and 10 ng of human pCMV-hAR-(1–660), pCMV-mAR-(1–640), p5M-hPR-B-(1–688), or pCMV-hGR-(1–550) with and without 1.5 μg of pSG5 (p5) empty vector (−), 0.5 μg of pSG5-MAGE (M), and 1 μg of pSG5-HA-p300 (P). E, pCMV-hAR-(1–660) (25 ng) was transfected into HeLa cells using Lipofectamine 2000 without siRNA (−) or with 10 nm p300 siRNA-1–4, nonspecific siRNA-3 (NS), and 0.1 μg of PSA-Enh-Luc. F, pCMV-hAR-(507–919) (50 ng) and pCMV-hAR-(1–503) (25 ng) were transfected in HeLa cells with 0.1 μg of PSA-Enh-Luc as above except with 5 nm p300 siRNAs and nonspecific siRNA-5 (NS5). Cells were incubated with and without 10 nm DHT.