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. 2003 Aug;23(16):5867–5881. doi: 10.1128/MCB.23.16.5867-5881.2003

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FIG. 6. — ER-dependent down regulation of the GR requires de novo protein synthesis and proteasome degradation. (A) CHX blocks ER mediated decrease in GR. MCF-7 cells were untreated (cytosol results, lane 1) or pretreated for 48 h with 10 nM E2 (lane 2) or 100 nM raloxifene (lane 3) in the presence or absence of 1 μg of CHX/ml (lanes 4 to 6). After 48 h, cells were untreated or treated with 1 nM DEX (nuclear results, lane 1) or ER ligands and DEX (lanes 2 and 3) for 12 h. Western blotting analysis was done with antibodies specific for GR, ER, and β-tubulin. (B) CHX blocks the inhibitory effect of E2 on GR transactivation. MCF-7 cells were untreated (lanes 1 and 7) or pretreated for 48 h with 10 nM E2 (lanes 3, 4, 9, and 10) or 100 nM raloxifene (lanes 5, 6, 11, and 12) in the presence or absence of 1 μg of CHX/ml (lanes 1 to 6). After 48 h, cells were treated with 1 nM DEX (lane 2 and 8) or ER ligands and DEX (lanes 4, 6, 10 and 12) for 12 h. Total RNA was harvested and analyzed by primer extension with specific primers for MMTV and 18S rRNA. PCR products were analyzed with 8% polyacrylamide denaturing gels and exposed to phosphorimager screens for further analysis. (C) The proteasome inhibitor MG132 blocks ER-mediated degradation of the GR. MCF-7 cells were untreated (lane 1) or pretreated for 48 h with 10 nM E2 (lane 2) or 100 nM raloxifene (lane 3) in the presence or absence of 0.5 μM MG132 (lanes 4 to 6). After 48 h, cells were treated with 1 nM DEX or ER ligands and DEX for 12 h. Western blotting analysis was done with antibodies specific for GR and ER. (D) The proteasome inhibitor MG132 blocks GR degradation, but not PR degradation. MCF-7 cells were untreated (lane 1) or pretreated for 48 h with 10 nM E2 (lane 2) or 100 nM raloxifene (lane 3) in the presence or absence of 0.5 μM MG132 (lanes 4 to 6). Nuclear extracts were prepared, and Western blotting analysis was done with antibodies specific for PR. (E) MG132 reverses the inhibitory effect of E2 on GR transactivation. MCF-7 cells were untreated (lanes 1 and 7) or pretreated for 48 h with 10 nM E2 (lanes 3, 4, 9, and 10) or 100 nM raloxifene (lanes 5, 6, 11, and 12) in the presence or absence of 0.5 μM MG132 (lanes 7 to 12). After 48 h, cells were treated with 1 nM DEX (lanes 2 and 8) or ER ligands and DEX (lanes 4, 6, 10, and 12) for 12 h. Total RNA was analyzed by RT-PCR with specific primers for MMTV and β₂-microglobulin. PCR products were analyzed with 8% polyacrylamide denaturing gels and exposed to phosphorimager screens. E, E2; R, raloxifene.

FIG. 6. — ER-dependent down regulation of the GR requires de novo protein synthesis and proteasome degradation. (A) CHX blocks ER mediated decrease in GR. MCF-7 cells were untreated (cytosol results, lane 1) or pretreated for 48 h with 10 nM E2 (lane 2) or 100 nM raloxifene (lane 3) in the presence or absence of 1 μg of CHX/ml (lanes 4 to 6). After 48 h, cells were untreated or treated with 1 nM DEX (nuclear results, lane 1) or ER ligands and DEX (lanes 2 and 3) for 12 h. Western blotting analysis was done with antibodies specific for GR, ER, and β-tubulin. (B) CHX blocks the inhibitory effect of E2 on GR transactivation. MCF-7 cells were untreated (lanes 1 and 7) or pretreated for 48 h with 10 nM E2 (lanes 3, 4, 9, and 10) or 100 nM raloxifene (lanes 5, 6, 11, and 12) in the presence or absence of 1 μg of CHX/ml (lanes 1 to 6). After 48 h, cells were treated with 1 nM DEX (lane 2 and 8) or ER ligands and DEX (lanes 4, 6, 10 and 12) for 12 h. Total RNA was harvested and analyzed by primer extension with specific primers for MMTV and 18S rRNA. PCR products were analyzed with 8% polyacrylamide denaturing gels and exposed to phosphorimager screens for further analysis. (C) The proteasome inhibitor MG132 blocks ER-mediated degradation of the GR. MCF-7 cells were untreated (lane 1) or pretreated for 48 h with 10 nM E2 (lane 2) or 100 nM raloxifene (lane 3) in the presence or absence of 0.5 μM MG132 (lanes 4 to 6). After 48 h, cells were treated with 1 nM DEX or ER ligands and DEX for 12 h. Western blotting analysis was done with antibodies specific for GR and ER. (D) The proteasome inhibitor MG132 blocks GR degradation, but not PR degradation. MCF-7 cells were untreated (lane 1) or pretreated for 48 h with 10 nM E2 (lane 2) or 100 nM raloxifene (lane 3) in the presence or absence of 0.5 μM MG132 (lanes 4 to 6). Nuclear extracts were prepared, and Western blotting analysis was done with antibodies specific for PR. (E) MG132 reverses the inhibitory effect of E2 on GR transactivation. MCF-7 cells were untreated (lanes 1 and 7) or pretreated for 48 h with 10 nM E2 (lanes 3, 4, 9, and 10) or 100 nM raloxifene (lanes 5, 6, 11, and 12) in the presence or absence of 0.5 μM MG132 (lanes 7 to 12). After 48 h, cells were treated with 1 nM DEX (lanes 2 and 8) or ER ligands and DEX (lanes 4, 6, 10, and 12) for 12 h. Total RNA was analyzed by RT-PCR with specific primers for MMTV and β₂-microglobulin. PCR products were analyzed with 8% polyacrylamide denaturing gels and exposed to phosphorimager screens. E, E2; R, raloxifene.