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Results: 5

1.
Fig. 3.

Fig. 3. From: Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.

ARNT selectivity for TACC3 is mediated by a conservative amino acid change within the TACC family. (A) Sequence alignment of TACC family CT20 residues. Residues mutated in D are indicated with numbering with respect to human TACC genes. (B) Ni-pulldown assay of His6-ARNT PAS-B with GST-TACC family fragments. (C) Ni-pulldown assay of His6-ARNT PAS-B with GST-TACC CT-swap and truncation fragments. (D) Ni-pulldown assay of His6-ARNT PAS-B with GST-TACC mutants.

Carrie L. Partch, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7739-7744.
2.
Fig. 4.

Fig. 4. From: Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.

Structural basis for TACC3 recruitment by ARNT PAS-B. (A) Close-up view of broadened residues within the 15N/1H HSQC spectra of 15N ARNT PAS-B titrated with natural abundance TACC3. (B) Significantly perturbed amide protons heat-mapped onto the ARNT PAS-B structure [Protein Data Bank (PDB) ID code 1XO0 (40)] as spheres according to degree of broadening. (C) HADDOCK model depicting simultaneous engagement of HIF-2α PAS-B (yellow) and the TACC3 C terminus (orange) by ARNT PAS-B (blue). (D) Close-up view of broadened residues within the 15N/1H HSQC spectra of 15N ARNT PAS-B with EDTA-derivatized TACC3 (M598C) chelated with either Ca2+ (gray) or Mn2+ (green). (E) Broadened residues mapped onto the ARNT PAS-B structure [PDB ID code 1XO0 (40)] in green, with dashed line showing approximately 28–35  distance from location of covalently attached EDTA moiety.

Carrie L. Partch, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7739-7744.
3.
Fig. 5.

Fig. 5. From: Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.

Mutations on ARNT PAS-B alter coactivator selectivity and HIF activation. (A) Close-up view of HADDOCK model illustrating ARNT PAS-B/TACC3 interface with ARNT PAS-B mutants shown in red. (B) Ni-pulldown assay of His6-ARNT PAS-B WT and mutants with GST-TACC3 or GST-TRIP230. (C) Densitometric analysis of bound GST-TACC3 (gray) or GST-TRIP230 (blue) with WT and mutant His6-ARNT PAS-B. (D) QPCR analysis of HIF target genes from 293T cells harvested 48 h after transfection with indicated plasmids. BNIP3, red; DEC2, blue; GLUT1, green; PGK1, orange. Data are normalized to individual target gene expression in the presence of WT FLAG-ARNT. Error bars, SD for n = 3 independent biological replicates.

Carrie L. Partch, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7739-7744.
4.
Fig. 2.

Fig. 2. From: Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.

A common interface in the C terminus of TACC3 mediates interaction with ARNT and other transcriptional regulators. (A) Ni-pulldown assay using purified His6-Gβ1-ARNT PAS-A or His6-ARNT PAS-B with soluble E. coli extracts containing overexpressed GST-tagged coactivators. (B) Illustration of various TACC3 truncations used in this study. Dashed line represents significance cutoff for coiled-coil analysis. (C) Ni-pulldown assay of His6-ARNT PAS-B with GST-TACC3 fragments. (D) Identification of CT20 mutants previously identified to disrupt TACC3 binding to FOG-1 (23). (E) Ni-pulldown assay of His6-ARNT PAS-B with mutant GST-TACC3 fragments. (F) QPCR analysis of HIF target genes from 293T cells harvested 48 h after transfection with indicated plasmids. Error bars, SD for n = 3 independent biological replicates.

Carrie L. Partch, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7739-7744.
5.
Fig. 1.

Fig. 1. From: Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.

TACC3 interacts with ARNT to regulate HIF transactivation. (A) Domain organization of HIF-1α, ARNT, and TACC3. Gray bars indicate domains of the bHLH-PAS proteins that mediate heterodimerization; dashed black bars indicate domains that interact with other transcriptional coregulators. (B) TACC3 coimmunoprecipitates with ARNT. HEK 293T cells were treated with 200 μM CoCl2 for 16 h prior to harvest. Antibodies to ARNT and a nonspecific (NS) control (GAL4 DNA-binding domain) were used to immunoprecipitate complexes from whole-cell lysates. Asterisk, nonspecific band. (C) Immunoblot analysis of whole-cell lysates for TACC3 expression from 293T cells harvested after CoCl2 (200 μM, 16 h) or transfected with shRNA vectors (48 h). (D) QPCR analysis of HIF target genes from 293T cells harvested 48 h after transfection with indicated plasmids. Expression levels were all normalized by dividing expression of the gene of interest by an internal standard cyclophilin B (GOI/CypB).

Carrie L. Partch, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7739-7744.

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