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doi: 10.1242/10.1242/jcs.00076

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A nucleolar targeting sequence in the Werner syndrome protein resides within residues 949-1092

Laboratory of Molecular Gerontology, National Institute on Aging, NIH 5600, Nathan Shock Drive, Baltimore, MD 21224, USA

View larger version (14K): [in a new window]   Fig. 1. (A) EGFP-WRN constructs used in this study. Numbers correspond to amino acid sequences. (B) Scheme of the functional domains of the WRN protein. HRDC, the helicase, RNAseD, C-terminal conserved region; RQC, RecQ helicase conserved region.

View larger version (45K): [in a new window]   Fig. 2. Intranuclear localization and expression of the EGFP-WRN fragments shown in Fig. 1A. HeLa cells were transfected with the following constructs: (A) EGFP-WRN (1-1432) (a-d); EGFP-WRN (54-946 +NLS) (e-h); EGFP-WRN (949-1432) (i-l); (B) EGFP-WRN (NLS) (a-d); EGFP-WRN (1072-1432) (e-h); and EGFP-WRN (949-1092 +NLS) (i-l). After transfection, the localization of each construct was analyzed by confocal microscopy. Two representative pictures of each construct are shown. (C) EGFP-WRN (949-1092 +NLS; green)-transfected cells were fixed, and probed with anti-B23 antibody (Texas Red). (D) Protein expression of the different EGFP-WRN constructs. Equal amounts of protein from EGFP-WRN-construct-transfected cell lysates were loaded in each lane. The asterisks correspond to the crossreactivity of the anti-GFP antibody. Arrows show the expression of the GFP fusion protein.

View larger version (80K): [in a new window]   Fig. 3. Intracellular localization and expression of EGFP-WRN fragments. (A) HeLa cells were transfected with the vector alone (EGFP) (a,b), EGFP-WRN (949-1092) (c,d) and EGFP-WRN (54-946) (e,f) constructs, and analyzed as described in Fig. 1A,B. One representative picture of each construct is shown. (B) Protein expression of EGFP-WRN constructs shown in panel A. After transfection the cells were analyzed as described in Fig. 2D.

View larger version (94K): [in a new window]   Fig. 4. Intranuclear localization of EGFP-WRN fragments in different human cell lines. U-2 OS (left) and AG11395 cells (right) were transfected with EGFP-WRN (a-d), EGFP-WRN (1072-1432) (e-h) and EGFP-WRN (949-1092 +NLS) (i-l) constructs as indicated on the left. After transfection the localization of each construct was analyzed by confocal microscopy.

View larger version (99K): [in a new window]   Fig. 5. Intranuclear localization of EGFP-WRN (human sequence) in a mouse cell line. B16F10 mouse cells were transfected with EGFP-WRN (a-d), EGFP-WRN (1072-1432) (e-h) and EGFP-WRN (949-1092 +NLS) (i-l) constructs as indicated on the left. After transfection the localization of each construct was analyzed by confocal microscopy.

View larger version (46K): [in a new window]   Fig. 6. The RQC domain deletion mutant of WRN localizes outside the nucleoli. (A) Schematic representation of the generation of the WRN853-1089 (I853L). Numbers correspond to amino acid sequences. (B) HeLa cells were transfected with EGFP-WRN853-1089 (I853L) and analyzed as described in Fig. 1A,B. One representative picture is shown.