0 10 Evaluation evaluation NN 11 13 of of IN 14 17 the the DT 18 22 role role NN 23 25 of of IN 26 32 ligand ligand NN 33 36 and and CC 37 44 thermal thermal JJ 45 55 activation activation NN 56 58 of of IN 59 67 specific specific JJ 68 71 DNA dna NN 72 79 binding binding NN 80 82 by by IN 83 85 in in FW 86 91 vitro vitro FW 92 103 synthesized synthesize VBN 104 109 human human JJ 110 124 glucocorticoid glucocorticoid NN 125 133 receptor receptor NN 133 134 . . . 136 138 We we PRP 139 143 have have VBP 144 148 used use VBN 149 150 a a DT 151 182 DNA-binding/immunoprecipitation DNA-binding/immunoprecipitation NNP 183 188 assay assay NN 189 191 to to TO 192 199 analyze analyze VB 200 203 the the DT 204 212 capacity capacity NN 213 215 of of IN 216 221 human human JJ 222 236 glucocorticoid glucocorticoid NN 237 245 receptor receptor NN 246 247 ( ( ( 247 250 hGR hGR NNP 250 251 ) ) ) 251 252 , , , 253 262 generated generate VBN 263 265 in in IN 266 272 rabbit rabbit NN 273 285 reticulocyte reticulocyte NN 286 293 lysates lysate NNS 293 294 , , , 295 297 to to TO 298 302 bind bind VB 303 306 DNA dna NN 306 307 . . . 308 310 In in FW 311 316 vitro vitro FW 317 327 translated translate VBN 328 331 hGR hGR NNP 332 335 was be VBD 336 353 indistinguishable indistinguishable JJ 354 358 from from IN 359 365 native native JJ 366 369 hGR hGR NNP 369 370 , , , 371 373 as as IN 374 384 determined determine VBN 385 387 by by IN 388 397 migration migration NN 398 400 on on IN 401 407 sodium sodium NN 408 415 dodecyl dodecyl NN 416 438 sulfate-polyacrylamide sulfate-polyacrylamide JJ 439 443 gels gel NNS 443 444 , , , 445 458 sedimentation sedimentation NN 459 461 on on IN 462 469 sucrose sucrose NN 470 477 density density NN 478 487 gradients gradient NNS 487 488 , , , 489 492 and and CC 493 503 reactivity reactivity NN 504 508 with with IN 509 520 antipeptide antipeptide NN 521 531 antibodies antibody NNS 532 541 generated generate VBN 542 549 against against IN 550 553 hGR hGR NNP 553 554 . . . 555 557 In in IN 558 566 addition addition NN 566 567 , , , 568 577 cell-free cell-free JJ 578 589 synthesized synthesize VBN 590 593 hGR hGR NNP 594 597 was be VBD 598 605 capable capable JJ 606 608 of of IN 609 617 specific specific JJ 618 625 binding binding NN 626 628 to to TO 629 643 glucocorticoid glucocorticoid NN 644 652 response response NN 653 660 element element NN 661 662 ( ( ( 662 677 GRE)-containing gre)-containing JJ 678 681 DNA DNA NNP 682 691 fragments fragment NNS 691 692 . . . 693 698 Using use VBG 699 703 this this DT 704 709 assay assay NN 710 716 system system NN 716 717 , , , 718 720 we we PRP 721 725 have have VBP 726 735 evaluated evaluate VBN 736 739 the the DT 740 753 contributions contribution NNS 754 756 of of IN 757 763 ligand ligand NN 764 771 binding binding NN 772 775 and and CC 776 780 heat heat NN 781 791 activation activation NN 792 794 to to TO 795 798 DNA DNA NNP 799 806 binding binding NN 807 809 by by IN 810 815 these these DT 816 830 glucocorticoid glucocorticoid NN 831 840 receptors receptor NNS 840 841 . . . 842 844 In in FW 845 850 vitro vitro FW 851 861 translated translate VBN 862 865 hGR hGR NNP 866 869 was be VBD 870 877 capable capable JJ 878 880 of of IN 881 890 selective selective JJ 891 894 DNA dna NN 895 902 binding binding NN 903 907 even even RB 908 910 in in IN 911 914 the the DT 915 922 absence absence NN 923 925 of of IN 926 940 glucocorticoid glucocorticoid NN 940 941 . . . 942 951 Treatment Treatment NNP 952 956 with with IN 957 970 dexamethasone dexamethasone NN 971 973 or or CC 974 977 the the DT 978 996 antiglucocorticoid antiglucocorticoid JJ 997 1002 RU486 RU486 NNP 1003 1006 had have VBD 1007 1009 no no DT 1010 1020 additional additional JJ 1021 1027 effect effect NN 1028 1030 on on IN 1031 1034 the the DT 1035 1046 DNA-binding dna-binding JJ 1047 1055 capacity capacity NN 1056 1060 when when WRB 1061 1069 receptor receptor NN 1070 1082 preparations preparation NNS 1083 1087 were be VBD 1088 1098 maintained maintain VBN 1099 1101 at at IN 1102 1103 0 0 CD 1104 1105 C C NNP 1106 1107 ( ( ( 1107 1109 no no DT 1110 1120 activation activation NN 1120 1121 ) ) ) 1121 1122 . . . 1123 1125 In in IN 1126 1134 contrast contrast NN 1134 1135 , , , 1136 1144 addition addition NN 1145 1147 of of IN 1148 1154 either either CC 1155 1161 ligand ligand NN 1162 1164 or or CC 1165 1175 antagonist antagonist NN 1176 1178 in in IN 1179 1190 combination combination NN 1191 1195 with with IN 1196 1197 a a DT 1198 1202 heat heat NN 1203 1213 activation activation NN 1214 1218 step step NN 1219 1227 promoted promote VBD 1228 1231 DNA dna NN 1232 1239 binding binding NN 1240 1242 by by IN 1243 1256 approximately approximately RB 1257 1263 3-fold 3-fold RB 1264 1268 over over IN 1269 1273 that that DT 1274 1276 of of IN 1277 1291 heat-activated heat-activated JJ 1292 1302 unliganded unliganded JJ 1303 1312 receptors receptor NNS 1312 1313 . . . 1314 1321 Agonist Agonist NNP 1322 1323 ( ( ( 1323 1336 dexamethasone dexamethasone NN 1336 1337 ) ) ) 1338 1341 was be VBD 1342 1350 slightly slightly RB 1351 1355 more more RBR 1356 1365 effective effective JJ 1366 1368 in in IN 1369 1379 supporting support VBG 1380 1388 specific specific JJ 1389 1392 DNA dna NN 1393 1400 binding binding NN 1401 1405 than than IN 1406 1416 antagonist antagonist NN 1417 1418 ( ( ( 1418 1423 RU486 ru486 NN 1423 1424 ) ) ) 1424 1425 . . . 1426 1429 DNA DNA NNP 1430 1437 binding binding NN 1438 1440 by by IN 1441 1443 in in FW 1444 1449 vitro vitro FW 1450 1461 synthesized synthesize VBN 1462 1464 GR GR NNP 1465 1468 was be VBD 1469 1476 blocked block VBN 1477 1479 by by IN 1480 1483 the the DT 1484 1492 addition addition NN 1493 1495 of of IN 1496 1502 sodium sodium NN 1503 1512 molybdate molybdate NN 1513 1515 to to TO 1516 1519 the the DT 1520 1528 receptor receptor NN 1529 1541 preparations preparation NNS 1542 1548 before before IN 1549 1556 steroid steroid NN 1557 1565 addition addition NN 1566 1569 and and CC 1570 1577 thermal thermal JJ 1578 1588 activation activation NN 1588 1589 . . . 1590 1598 Addition addition NN 1599 1601 of of IN 1602 1605 KCl KCl NNP 1606 1614 resulted result VBD 1615 1617 in in IN 1618 1622 less less JJR 1623 1626 DNA dna NN 1627 1634 binding binding NN 1635 1641 either either CC 1642 1645 due due JJ 1646 1648 to to TO 1649 1657 blockage blockage NN 1658 1660 of of IN 1661 1673 DNA-receptor DNA-receptor NNP 1674 1681 complex complex NN 1682 1691 formation formation NN 1692 1694 or or CC 1695 1705 disruption disruption NN 1706 1708 of of IN 1709 1712 the the DT 1713 1722 complexes complex NNS 1722 1723 . . . 1724 1727 The the DT 1728 1739 specificity specificity NN 1740 1742 of of IN 1743 1746 DNA DNA NNP 1747 1754 binding binding NN 1755 1757 by by IN 1758 1767 cell-free cell-free JJ 1768 1779 synthesized synthesize VBN 1780 1783 hGR hGR NNP 1784 1787 was be VBD 1788 1796 analyzed analyze VBN 1797 1804 further further RB 1805 1807 by by IN 1808 1817 examining examine VBG 1818 1821 the the DT 1822 1831 abilities ability NNS 1832 1834 of of IN 1835 1842 various various JJ 1843 1847 DNAs dna NNS 1848 1850 to to TO 1851 1858 compete compete VB 1859 1862 for for IN 1863 1870 binding bind VBG 1871 1873 to to TO 1874 1875 a a DT 1876 1885 naturally naturally RB 1886 1895 occurring occur VBG 1896 1899 GRE GRE NNP 1900 1905 found find VBN 1906 1908 in in IN 1909 1912 the the DT 1913 1918 mouse mouse NN 1919 1926 mammary mammary JJ 1927 1932 tumor tumor NN 1933 1943 virus-long virus-long JJ 1944 1952 terminal terminal JJ 1953 1959 repeat repeat NN 1959 1960 . . . 1961 1977 Oligonucleotides oligonucleotide NNS 1978 1988 containing contain VBG 1989 1992 the the DT 1993 2002 consensus consensus NN 2003 2006 GRE GRE NNP 2007 2011 were be VBD 2012 2015 the the DT 2016 2020 most most RBS 2021 2030 efficient efficient JJ 2031 2042 competitors competitor NNS 2042 2043 , , , 2044 2047 and and CC 2048 2057 fragments fragment NNS 2058 2068 containing contain VBG 2069 2079 regulatory regulatory JJ 2080 2089 sequences sequence NNS 2090 2094 from from IN 2095 2121 glucocorticoid-repressible glucocorticoid-repressible JJ 2122 2127 genes gene NNS 2128 2132 were be VBD 2133 2141 somewhat somewhat RB 2142 2153 competitive competitive JJ 2153 2154 , , , 2155 2162 whereas whereas IN 2163 2169 single single JJ 2170 2178 stranded stranded JJ 2179 2195 oligonucleotides oligonucleotide NNS 2196 2200 were be VBD 2201 2207 unable unable JJ 2208 2210 to to TO 2211 2218 compete compete VB 2219 2222 for for IN 2223 2228 mouse mouse NN 2229 2236 mammary mammary JJ 2237 2242 tumor tumor NN 2243 2253 virus-long virus-long JJ 2254 2262 terminal terminal JJ 2263 2269 repeat repeat NN 2270 2273 DNA dna NN 2274 2281 binding binding NN 2281 2282 , , , 2283 2289 except except IN 2290 2294 when when WRB 2295 2305 competitor competitor NN 2306 2309 was be VBD 2310 2317 present present JJ 2318 2320 at at IN 2321 2330 extremely extremely RB 2331 2335 high high JJ 2336 2350 concentrations concentration NNS 2350 2351 . . . 2352 2360 Together together RB 2361 2366 these these DT 2367 2374 studies study NNS 2375 2383 indicate indicate VBP 2384 2388 that that IN 2389 2392 hGR hGR NNP 2393 2404 synthesized synthesize VBN 2405 2407 in in IN 2408 2414 rabbit rabbit NN 2415 2427 reticulocyte reticulocyte NN 2428 2435 lysates lysate NNS 2436 2444 displays display VBZ 2445 2449 many many JJ 2450 2452 of of IN 2453 2456 the the DT 2457 2461 same same JJ 2462 2472 properties property NNS 2472 2473 , , , 2474 2483 including include VBG 2484 2496 GRE-specific gre-specific JJ 2497 2500 DNA dna NN 2501 2508 binding binding NN 2508 2509 , , , 2510 2518 observed observe VBN 2519 2522 for for IN 2523 2537 glucocorticoid glucocorticoid NN 2538 2546 receptor receptor NN 2547 2554 present present JJ 2555 2557 in in IN 2558 2567 cytosolic cytosolic JJ 2568 2576 extracts extract NNS 2577 2579 of of IN 2580 2589 mammalian mammalian JJ 2590 2595 cells cell NNS 2596 2599 and and CC 2600 2607 tissues tissue NNS 2607 2608 . . . 2609 2621 Similarities similarity NNS 2622 2629 between between IN 2630 2633 the the DT 2634 2641 effects effect NNS 2642 2644 of of IN 2645 2658 dexamethasone dexamethasone NN 2659 2662 and and CC 2663 2668 RU486 RU486 NNP 2669 2676 suggest suggest VBP 2677 2681 that that IN 2682 2685 the the DT 2686 2704 antiglucocorticoid antiglucocorticoid JJ 2705 2715 properties property NNS 2716 2718 of of IN 2719 2724 RU486 RU486 NNP 2725 2727 do do VBP 2728 2731 not not RB 2732 2737 occur occur VB 2738 2740 at at IN 2741 2744 the the DT 2745 2750 level level NN 2751 2753 of of IN 2754 2762 specific specific JJ 2763 2766 DNA dna NN 2767 2774 binding binding NN 2774 2775 . . .