Journal: Nuclear Receptor

Article Title: Thyroid hormone receptor binding to DNA and T 3 -dependent transcriptional activation are inhibited by uremic toxins

doi: 10.1186/1478-1336-3-1

Figure Lengend Snippet: Uremic plasma reduces hTRβ1-hRXRα complex formation on DR-4. Gel Shift experiments were performed using in vitro translated [ 35 S] hTRβ1, cold hRXRα and DR-4. [ 35 S] hTRβ1 was treated with T 3 10 -7 M for 30 min at 4°C and then incubated without (Control – lane 1) or with increasing volumes (0.5, 1.0, 2.0 μL) of normal (lanes 2–4) or uremic (lanes 5–7) plasma for 30 min at 4°C. Cold DR-4 type TRE (5'- AGCT TC AGGTCA CAGG AGGTCA GAG - 3'), cold hRXRα, and nonspecific DNA poly (dIdC) were subsequently added and incubated for 20 min.

Article Snippet: Following plasma exposure, the nuclear receptors were incubated for another 20 min at room temperature (20–30°C) in a solution containing 2 μg non-specific DNA poly (dIdC) (Pharmacia LKB, Piscataway, NJ), cold specific response element (10 ng/reaction), nonradioactive RXRα and a binding buffer in a 20 μL reaction as previously described [ ].

Techniques: Clinical Proteomics, Gel Shift, In Vitro, Incubation, Control

Journal: Nuclear Receptor

Article Title: Thyroid hormone receptor binding to DNA and T 3 -dependent transcriptional activation are inhibited by uremic toxins

doi: 10.1186/1478-1336-3-1

Figure Lengend Snippet: Hemodialysis reduces the inhibitory effect of uremic plasma on hTRβ1-hRXRα binding to DR-4. [ 35 S] hTRβ1 was pre-incubated without (Control – lane 1) or with increasing volumes (0.5, 1.0, 2.0 μL) of normal (lane 2–4) or uremic plasma (patient 1) collected before (pre-HD – lanes 5–7) or after hemodialysis (pos-HD – lanes 8–10) for 30 min at 4°C. Cold DR-4 type TRE, cold hRXRα, and nonspecific DNA poly (dIdC) were subsequently added and incubated for 20 min.

Article Snippet: Following plasma exposure, the nuclear receptors were incubated for another 20 min at room temperature (20–30°C) in a solution containing 2 μg non-specific DNA poly (dIdC) (Pharmacia LKB, Piscataway, NJ), cold specific response element (10 ng/reaction), nonradioactive RXRα and a binding buffer in a 20 μL reaction as previously described [ ].

Techniques: Clinical Proteomics, Binding Assay, Incubation, Control

Journal: Nuclear Receptor

Article Title: Thyroid hormone receptor binding to DNA and T 3 -dependent transcriptional activation are inhibited by uremic toxins

doi: 10.1186/1478-1336-3-1

Figure Lengend Snippet: Uremic plasma inhibits VDR-RXRα-DR-3 complex formation and hemodialysis reduces this effect. Gel Shift experiments were performed using in vitro translated [ 35 S] hVDR and cold hRXRα. [ 35 S] hVDR was incubated with VD 3 vitamin for 30 min at 4°C and then without (Control – lane 1) or with increasing volumes (0.5, 1.0, 2.0 μL) of normal (lanes 2–4) or uremic plasma, collected before (pre-HD – lanes 5–7) and after hemodialysis (pos-HD – lanes 8–10) from patient 1, for 30 min. Cold DR-3 type VDRE (5'- AGCT TC AGGTCA AGG AGGTCA GAG - 3'), cold hRXRα, and nonspecific DNA poly (dIdC) were subsequently added and incubated for 20 min.

Article Snippet: Following plasma exposure, the nuclear receptors were incubated for another 20 min at room temperature (20–30°C) in a solution containing 2 μg non-specific DNA poly (dIdC) (Pharmacia LKB, Piscataway, NJ), cold specific response element (10 ng/reaction), nonradioactive RXRα and a binding buffer in a 20 μL reaction as previously described [ ].

Techniques: Clinical Proteomics, Gel Shift, In Vitro, Incubation, Control

Journal: Nuclear Receptor

Article Title: Thyroid hormone receptor binding to DNA and T 3 -dependent transcriptional activation are inhibited by uremic toxins

doi: 10.1186/1478-1336-3-1

Figure Lengend Snippet: Uremic plasma does not decrease PPARγ-RXRα-DR-1 complex formation. Gel Shift experiments were performed using in vitro translated [ 35 S] PPARγ and cold hRXRα. [ 35 S] PPARγ was incubated without (Control – lane 1) or with increasing volumes (0.5, 1.0, 2.0 μL) of normal (lanes 2–4) or uremic (lanes 5–7) plasma for 30 minutes. Cold DR-1 type TRE (5'- AGCT TC AGGTCA G AGGTCA GAG - 3'), cold hRXRα, and nonspecific DNA poly (dIdC) were subsequently added and the reaction was incubated for 20 min.

Article Snippet: Following plasma exposure, the nuclear receptors were incubated for another 20 min at room temperature (20–30°C) in a solution containing 2 μg non-specific DNA poly (dIdC) (Pharmacia LKB, Piscataway, NJ), cold specific response element (10 ng/reaction), nonradioactive RXRα and a binding buffer in a 20 μL reaction as previously described [ ].

Techniques: Clinical Proteomics, Gel Shift, In Vitro, Incubation, Control

Journal: Nuclear Receptor

Article Title: Thyroid hormone receptor binding to DNA and T 3 -dependent transcriptional activation are inhibited by uremic toxins

doi: 10.1186/1478-1336-3-1

Figure Lengend Snippet: Heating the uremic plasma does not diminish the inhibition of hTRβ1-hRXRα binding to DR-4. Gel Shift experiments were performed using in vitro translated cold hTRβ1, cold hRXRα and [ 32 P]DR-4. Cold hTRβ1 was pre-incubated with normal or uremic plasma, heated or not, and in the presence or absence of phosphatase inhibitor for 30 min at 4°C. [ 32 P] DR-4 type TRE, cold hRXRα, and nonspecific DNA poly (dIdC) were subsequently added and the reaction was incubated for 20 min.

Article Snippet: Following plasma exposure, the nuclear receptors were incubated for another 20 min at room temperature (20–30°C) in a solution containing 2 μg non-specific DNA poly (dIdC) (Pharmacia LKB, Piscataway, NJ), cold specific response element (10 ng/reaction), nonradioactive RXRα and a binding buffer in a 20 μL reaction as previously described [ ].

Techniques: Clinical Proteomics, Inhibition, Binding Assay, Gel Shift, In Vitro, Incubation