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1.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 7. Effect of NES placement outside the DNA-binding domain of STAT1. STAT1–NES–GFP was transfected into U3A cells either untreated (a and b) or treated with IFN-γ (c and d) for 30 min in the presence (right panels) or absence (left panels) of leptomycin B (LMB), and localization was determined by fluorescent microscopy.

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
2.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 5. NES location in the crystal structure of tyrosine-phosphorylated STAT1 dimer (residues 136–710) bound to DNA (Chen et al., 1998).The DNA is oriented perpendicular to the paper. The NES (residues 400–409) (yellow) is indicated on a STAT1 monomer (blue). The ribbon diagram was prepared using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1994) from coordinates posted at the web site (http://www.Rockefeller.edu/Kuriyan). Secondary structural elements of the crystal structure were checked with PROMOTIF (Hutchinson and Thornton, 1996).

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
3.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 2. Leptomycin B inhibits STAT1 nuclear export. (A) U3A cells expressing STAT1–GFP were pre-treated for 30 min with cyclo heximide (all panels) and leptomycin B (LMB) (right panels) and then pulse-treated with IFN-γ for 30 min. The cellular localization of STAT1–GFP was visualized by fluorescent microscopy. Time after IFN-γ treatment is indicated to the left of the panels. (B) Cells from each treatment condition in (A) were collected and immunoprecipitated with anti-STAT1 antibody. Western blots were performed with anti-STAT1 phosphotyrosine antibody (anti-STAT1 pY) (upper panel) or anti-STAT1 antibody (lower panel).

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
4.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 8. Effect of addition of a characterized NLS to STAT1. (A) HT1080 cells were transfected with either STAT1–GFP (a) or STAT1–NLS–GFP (b) and localization determined in untreated cells by fluorescent microscopy. (B) HT1080 cells stably expressing STAT1–NLS–GFP were pre-treated with cycloheximide (all lanes) and leptomycin B (LMB) (lanes 3 and 5) for 30 min. Cells were left untreated, or treated with IFN-γ alone or with IFN-γ and pervanadate (PV). Proteins were immunoprecipitated from lysates with anti-STAT1 antibody, and western blots were performed with anti-STAT1 phosphotyrosine antibody (anti-STAT1 pY) (upper panel), anti-STAT1 antibody (middle panel) or anti-GFP antibody (lower panel).

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
5.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 1. STAT1–GFP is activated by IFN-γ treatment. (A) U3A cells expressing STAT1–GFP were untreated (a) or treated (b) with IFN-γ for 30 min. STAT1–GFP cellular localization was examined by fluorescent microscopy. (B) U3A cells (lanes 1 and 2) or U3A cells expressing STAT1–GFP (lanes 3 and 4) were untreated or treated with IFN-γ for 30 min. Proteins were immunoprecipitated with anti-STAT1 antibody and western blots performed with anti-STAT1 phosphotyrosine antibody (anti-STAT1 pY) (upper panel) or anti-GFP antibody (lower panel). (C) Nuclear extracts were prepared from U3A cells expressing STAT1–GFP and were untreated or treated for 30 min with IFN-γ. EMSA was performed with the IRF-1 GAS probe. Anti-STAT1 antibody was incubated with extracts prior to addition of probe.

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
6.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 3. Ran-dependent binding of CRM1 to STAT1. (AIn vitro transcribed/translated [35S]methionine-labeled CRM1 was incubated with either GST (lane 1), GST–STAT1 (lanes 2–4) or GST–NESPKI (lanes 5–7) fusion proteins bound to glutathione beads in the presence or absence of Ran. HIV Rev NES peptide (NESRev) competitor was added to demonstrate specificity (lanes 4 and 7). The lower panel shows that equal amounts of CRM1 were added to the assay. (B) The CRM1 binding assay was performed in the presence of Ran with GST–STAT1 fusion proteins corresponding to residues 1–750, 1–526, 1–448, 1–436, 1–369 or 1–315 of STAT1.

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
7.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 6. Localization of STAT1 DNA-binding mutant. (A) The DNA-binding mutant STAT1–GFP EE428/429SA was expressed in U3A cells and localized by fluorescent microscopy. Cells were untreated (a and b) or treated (c and d) with IFN-γ for 30 min in the presence (right panels) or absence (left panels) of leptomycin B (LMB). (B) U3A cells (lanes 1 and 2) or U3A cells transfected with STAT1–GFP EE428/429SA (lanes 3 and 4) were untreated or treated with IFN-γ for 30 min. Proteins were immunoprecipitated from cell lysates with anti-GFP antibody, and western blots were performed with anti-STAT1 phosphotyrosine antibody (anti-STAT1 pY) (upper panel) or anti-STAT1 antibody (lower panel).

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
8.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 9. (A) Illustration of STAT1 cellular localization. Following ligand binding to receptors, latent cytoplasmic STAT1 is tyrosine phosphorylated by JAKs and phosphorylation causes dimerization (1). The dimers are imported into the nucleus where they bind to a DNA target site in the promoters of responsive genes (2). The NES of STAT1 is masked when STAT1 dimers are bound to DNA. A nuclear tyrosine phosphatase can dephosphorylate STAT1, releasing it from DNA (3). The NES of STAT1 is now accessible to CRM1, resulting in export to the cytoplasm (4). (B) Alignment of STAT1 NES with putative NES sequences of other STAT family members.

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.
9.

Figure. From: Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Fig. 4. Identification of the STAT1 NES. (A) Localization of GFP alone (a and b) or STAT1–GFP fusion constructs containing STAT1 residues 368–436 (c and d), 377–413 (e and f), 392–413 (g and h) or 424–439 (i and j) was determined in U3A cells in the presence (right panels) or absence (left panels) of leptomycin B (LMB). (B) Alignment of the STAT1 NES with characterized NES sequences from cyclin B, protein kinase A inhibitor-α (PKIα), mitogen-activated protein kinase kinase (MAPKK), the Rev protein of HIV (HIV-Rev), p53 and the Rex protein of the human T-cell leukemia virus (HTLV-REX). This alignment reveals spatial conservation of critical hydrophobic amino acids (boxed). (C) Localization of STAT1–GFP fusion proteins with point mutations in the NES was evaluated by fluorescent microscopy. Point mutations (a) Val374 to glutamate,(b) Ile425 to glutamine and (f) Leu409 to alanine were made in the context of STAT1 residues 368–436 fused to GFP and expressed in U3A cells. Mutations (c) Leu400 to alanine, (d) Phe404 to serine and (e) Leu407 to alanine were made in the context of STAT1 residues 392–413 fused to GFP.

Kevin M. McBride, et al. EMBO J. 2000 November 15;19(22):6196-6206.

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