Figure 5. Both IDs Are Capable of Functionally Interacting with the TR on Positive TREs CV-1 cells were cotransfected with 1.7 μg of LYS (A) PAL (B), or DR+4 (C) reporter plasmid and 80 ng of pKCR2-TR and/or 80 ng of pKCR2 and/or 80 ng of pKCR2-hNCoRI deletion construct. The data are quantified as fold repression (mean ± se). D, CV-1 cells were transfected with GFP alone (mock) or the indicated NCoR construct and subjected to fluorescence microscopy. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 4. NCoR Prefers to Interact with the TR A, GST-TR was used to pull down radiolabeled NCoR or SMRT. I, 20% input; G, GST alone. The concentration of T<sub>3</sub> used was 10<sup>−6</sup>m. B, EMSA was performed using in vitro translated proteins and a radiolabeled DR + 4 probe. The resolved complexes are indicated. C, CV-1 cells were transfected with 1.7 μg of the UAS reporter with 80 ng of the indicated GAL4 construct and 80 ng of the indicated VP-16 vector. The data are quantified as fold activation in the presence of empty VP-16 expression vector. D, CV-1 cells were transfected with increasing amounts of either NCoRI or SMRTI on the DR+4 reporter. The data are shown as fold repression in the presence of TR alone. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 3. Sequence Alignments of the NCoR ID Sequences Differ from SMRT and Each Other, but Are Conserved across Species A, The amino acid sequences of ID I and ID II from human NCoRI were aligned with the hSMRT AA sequence and the area of identity was shown. B, The ID I and ID II sequences from hNCoRI were aligned with the analogous sequences from mNCoR (6 ) and each other. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 2. NCoR and NCoRI Have Separate Actions in CV-1 Cells CV-1 cells were cotransfected with 1.7 μg of the indicated reporter construct and 80 ng of pKCR2-TR with either 330 ng of NCoR, NCoRI, or empty expression vector. The data are quantified as relative luciferase activity (mean ± se) with basal activity in the absence of TR set as 1. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 1. hNCoRI Deletion Constructs A, mNCoR and hNCoRI are aligned schematically. Amino acid numbering is per mNCoR sequence. hNCoRI has 93% homology when compared with the comparable region of mNCoR. B, hNCoRI constructs lacking ID I and/or ID II were constructed, as described in Materials and Methods, and placed into the expression vector pKCR2. Amino acids corresponding to NCoRI sequence are identified with the corresponding sequence designations of mNCoR noted in parentheses. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 6. hNCoRI Preferentially Binds TR Homodimer A gel mobility shift assay was carried out using in vitro translated proteins from rabbit reticulocyte lysate, except where indicated, and a labeled DR+4 oligonucleotide probe. After a 20-min room temperature incubation, 1 μl anti-RXRα (A), anti-TRb (B), or anti-C/EBP (B) antisera were added. Incubations were then carried out for an additional 20 min at room temperature. C, A gel-mobility shift assay was carried out using 20 ng of GST-NCoR with increasing amounts of TRβ1 (shown as microliters of reticulocyte lysate) with the amount of RXR held constant in the presence of a labeled DR+4 oligonucleotide probe. D and E, CV-1 cells were transfected with the indicated constructs. The data are expressed as relative luciferase activity with the activity of GAL4-TR set as 1. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 6. hNCoRI Preferentially Binds TR Homodimer A gel mobility shift assay was carried out using in vitro translated proteins from rabbit reticulocyte lysate, except where indicated, and a labeled DR+4 oligonucleotide probe. After a 20-min room temperature incubation, 1 μl anti-RXRα (A), anti-TRb (B), or anti-C/EBP (B) antisera were added. Incubations were then carried out for an additional 20 min at room temperature. C, A gel-mobility shift assay was carried out using 20 ng of GST-NCoR with increasing amounts of TRβ1 (shown as microliters of reticulocyte lysate) with the amount of RXR held constant in the presence of a labeled DR+4 oligonucleotide probe. D and E, CV-1 cells were transfected with the indicated constructs. The data are expressed as relative luciferase activity with the activity of GAL4-TR set as 1. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 7. Both IDs Mediate Binding of NCoR to TR on DNA Response Elements Gel mobility shift assays were carried out using 3–4 μl of in vitro translated proteins from rabbit reticulocyte lysate and <sup>32</sup>P-radiolabeled LYS (A) PAL (B), or DR+4 (C) oligonucleotide probes. The labeled probe was incubated with in vitro translated TRb1 and hNCoRI deletion construct, or unprogrammed reticulocyte lysate as indicated. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 8. NCoR IDs Bind TR Homodimer The DR+4 probe was incubated with TRβ1 and RXRα and/or hNCoRI deletion construct, in the absence (A) or presence (B) of 10 nm T<sub>3</sub>. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society
Figure 9. hNCoRI Deletion Constructs Enhance Ligand-Independent Activation on a Negative TRE TRH-pA3Luc (1.7 μg) was cotransfected with 80 ng pKCR2-TR and/or 80 ng pKCR2 alone and/or pKCR2-hNCoRI deletion construct. Each well was also transfected with 20 ng pCMV-βGAL to control for transfection efficiency. The data are quantified as relative fold activation, where 1 represents activation in the presence of pKCR2-TR, but not pKCR2-hNCoRI, corrected for β-galactosidase activity. From: Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements Mol Endocrinol. 1998;12(10):1567-1581. doi:10.1210/mend.12.10.0188 Mol Endocrinol | Copyright © 1998 by The Endocrine Society