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Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8578, Japan

	Abstract

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The LBP-1 family consists of four proteins, which act as transcription factors in the formation of dimers with a member of this family. LBP-1a and LBP-1b are splicing variants from one gene, and LBP-1c and LBP-1d also arise from the alternative splicing of another gene. Investigation of subcellular localization of LBP-1 proteins fused to YFP revealed that the LBP-1b was localized in the nucleus, whereas LBP-1a and LBP-1c were exclusively localized in the cytosol. The peptide of 36 amino acids encoded by exon 6, a specific exon used only for LBP-1b, possessed the function of a nuclear localization signal (NLS). Nuclear localization of LBP-1a and LBP-1c occurred when LBP-1b was co-expressed, suggesting that heterodimerization of LBP-1a and LBP-1c with LBP-1b is important for their nuclear transport. Transiently expressed LBP-1 proteins in COS-7 cells formed speckles in the nucleus. Most speckles overlapped with the PML body. The activity of LBP-1a for accumulation in the PML body was mapped in the N-terminal region.

	Introduction

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Proteins homologous to LBP-1 proteins constitute a growing family of transcription factors. This family was classified into two large groups, the LBP family and the GRH family of proteins, on the basis of sequence similarity. The first group includes LBP-1 termed as leader-binding protein-1 that binds to the human immunodeficiency virus type 1 (HIV-1) LTR and represses Tat-induced HIV-1 transcription (Garcia et al. 1987; Jones et al. 1988). LBP-1c (also called CP2, LSF, UBP-1 or SEF), the most investigated member of the LBP-1 proteins, was initially defined as a CCAAT-binding protein to the mouse -globin promoter (Lim et al. 1992) or a transcriptional activator of the SV40 late promoter (Huang et al. 1990). Later studies showed that there are two distinct LBP-1 genes, each of which encodes two alternatively spliced variants, so that LBP-1a (also referred to as NF2d9) and LBP-1b arise from one gene, and LBP-1c and LBP-1d arise from the other (Yoon et al. 1994). Since LBP-1d lacks a part of the GRH/NTF-1/Elf-1 homology which is required for DNA binding, it cannot bind to DNA (Yoon et al. 1994). It has been shown that LBP-1a, LBP-1b and LBP-1c are ubiquitously expressed (Swendeman et al. 1994; Ramamurthy et al. 2001) and form homodimers or heterodimers with other LBP-1 proteins (Yoon et al. 1994; Zhong et al. 1994). Several reports have shown that LBP-1c forms a tetramer on binding to DNA (Shirra & Hansen 1998). The multimeric LBP-1 proteins recognize a hyphenated DNA sequence composed of 4-base motifs separated by a 6-base (or rarely 5-base) linker, CNRG-N6-CNR(G/C) (Lim et al. 1993; Yoon et al. 1994), although the DNA-binding sequence for LBP-1 proteins was not limited to this consensus. Analysis of the DNA binding region of LBP-1 proteins has shown that it is unlike any established DNA binding motifs, and phosphorylation of a serine residue in the domain by mitogen activated protein kinases enhanced the DNA binding activity (Volker et al. 1997). LBP-1 proteins are widely expressed and important for diverse cellular, viral and developmental functions, such as globin gene expression (Lim et al. 1992), lens-specific expression of A-crystallin gene (Murata et al. 1998), HIV-1 virus expression (Garcia et al. 1987; Jones et al. 1988; Yoon et al. 1994), gene regulation for male-specific Cyp 2d-9 (Sueyoshi et al. 1995), and T-cell proliferation (Casolaro et al. 2000). Mice with loss of LBP-1c expression were generated by homologous recombination (Ramamurthy et al. 2001). The mice displayed no defects in growth, behavior, fertility or development, and showed no phenotype in hematopoietic differentiation, globin gene expression or immunological responses to T- and B-cell mitogenic stimulation. These observations were attributed to functional compensation of LBP-1a for the loss of LBP-1c, strongly suggesting redundancy for all functions of the proteins. LBP-9 shows 83% sequence similarity to LBP-1b and has been shown to be a suppressor factor inhibiting the stimulation effect of LBP-1b (Huang & Miller 2000). Although expression patterns of LBP-9 were not examined in animal tissues, analysis of expression in several cultured cell lines suggests limited expression of LBP-9 in the tissues.

The second group consists of mammalian homologs of Drosophila grainyhead (dGRH), which plays important roles in early fly development. dGRH shows 42% similarity to mouse LBP-1c. In particular, a region of dGRH which appears to be required for DNA binding, shows a high sequence similarity to LBP-1c. MGR (also named LBP-32), mammalian counterpart of dGRH, Brother-of-MGR (BOM), and Sister-of-MGR (SOM), have been identified as members of this group (Wilanowski et al. 2002; Ting et al. 2003). These proteins show a highly restricted temporal pattern of expression in early development and limited expression among adult tissues, and could not heterodimerize with LBP-1 proteins (Wilanowski et al. 2002). Like LBP-1 proteins, several splicing variants arising from the genes for MGR and SOM have been reported (Wilanowski et al. 2002; Ting et al. 2003).

In the course of our study that investigated the role of LBP-1 proteins in the expression of a certain gene, we unexpectedly found that transiently expressed LBP-1a and LBP-1c could not enhance relevant reporter activity, although LBP-1b caused strong activation. In this paper, we describe differences in subcellular localization between LBP-1 proteins and the mechanism of their nuclear localization. LBP-1b contains a nuclear localization signal that arises from the unique exon 6 for LBP-1b. It is suggested that nuclear localization of LBP-1a and LBP-1c occurs by heterodimerization with LBP-1b. Subnuclear localization of LBP-1 proteins and their targeting to the PML body are also described.

	Results

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Subcellular localization of LBP-1 proteins

We have examined subcellular localization of LBP-1 proteins in the cells. Chimeric proteins of LBP-1 proteins fused to YFP (yellow fluorescent protein) were expressed in HepG2, HeLa and COS-7 cells and subcellular localization of the proteins was observed. As shown in Fig. 1, the YFP-LBP-1b was localized exclusively in the nucleus of the cells, and LBP-1b-YFP also localized in the nucleus of HepG2 cells. On the other hand, LBP-1a and LBP-1c exhibited cytosolic localization, and no signal was detected in the nucleus. Treatment of the cells with leptomycin B did not affect the subcellular localization of the proteins, suggesting no shuttling of these proteins between nucleus and cytosol (data not shown). LBP-1b expressed in COS-7 cells formed speckles in the nucleus.

View larger version (47K): [in this window] [in a new window]   Figure 1  Subcellular localization of LBP-1 proteins. YFP, YFP-LBP-1 and LBP-1-YFP chimeric proteins were transiently expressed in HepG2 cells, and YFP and YFP-LBP-1 proteins were also expressed in HeLa or COS-7 cells by DNA transfection. 24 h (COS-7) or 44 h (HepG2 and HeLa) after transfection, cells were fixed with 4% paraformaldehyde. Fluorescence of YFP was detected with an Olympus BX50 fluorescence microscope. Nuclei are labeled with DAPI. Scale bars, 20 µm.

LBP-1b differs from LBP-1a in that the former contains 36 amino acids arising from the unique exon 6. Therefore, we analyzed activity of the peptide for nuclear localization signal (NLS) by connecting this peptide to several YFP-fusion proteins. As shown in Fig. 2, the chimeric protein by which the peptide was attached to the N-terminal of YFP was localized in the nucleus of HeLa and COS-7 cells, strongly suggesting the presence of NLS in the peptide. Furthermore, a chimeric protein (NLS-YFP-LBP-1a) with the peptide attached to the N-terminal of YFP-LBP-1a was expressed in the cells as shown in Fig. 2. The chimeric protein also exhibited nuclear localization, confirming the NLS activity of the peptide. In the peptide, two clusters of basic amino acids were localized (Fig. 3). Three dibasic pairs of amino acids in the NLS were separately mutated to alanines and subcellular localization of the mutants was examined as shown in Fig. 3. When mutations were introduced in NLS-YFP, the three mutants were localized in the cytosol in addition to the nucleus, and the distribution was quite similar to that of YFP (see Figs 1 and 2). This increased cytosolic localization by mutation was observed both in HeLa and COS-7 cells. The same mutations were introduced into YFP-LBP-1b, and subcellular localization of the mutated proteins was examined. As shown in Fig. 3, the three mutants clearly showed cytosolic localization and very little or no signal of nuclear localization was found. Taken together, these results indicate that the basic amino acids in the two clusters separated by 11 amino acids were important for nuclear localization of LBP-1b, and suggest that the NLS of LBP-1b may be of a bipartite type.

View larger version (31K): [in this window] [in a new window]   Figure 2  Nuclear localization signal of LBP-1b. Nuclear localization of YFP-fusions with a peptide of NLS was examined. A DNA fragment for NLS was amplified by PCR and inserted into 5' to EYFP-N1 or pYFP-LBP-1a. Structure of YFP chimeric proteins is schematically represented on the left. The YFP chimeric proteins and original YFP were transiently expressed in HeLa or COS-7 cells. Cells were fixed with 4% paraformaldehyde, and fluorescence of YFP was observed with an Olympus BX50 fluorescence microscope. Nuclei are labeled with DAPI. Scale bars, 20 µm.

View larger version (54K): [in this window] [in a new window]   Figure 3  Mutational analysis of NLS of LBP-1b. Amino acid sequence of the peptide derived from exon 6 of the LBP-1b gene and mutated sequences are shown at the top. Dibasic pairs of amino acids are shown in red letters. Numbers of amino acids begin with the first amino acid encoded by exon 6. Structures of NLS-YFP, YFP-LBP-1b and their mutated derivatives are schematically shown on the left. Red and black vertical bars show wild and mutated NLS, respectively. Three dibasic pairs, K16-R17, K20-R21 and K33-R34, in the NLS in the NLS-YFP and YFP-LBP-1b were mutated to alanines, and nuclear localization of the mutants was examined by expressing them in HeLa or COS-7 cells. Cells were fixed with 4% paraformaldehyde, and fluorescence of YFP was observed with an Olympus BX50 fluorescence microscope. Nuclei are labeled with DAPI. Scale bars, 20 µm.

As reported, LBP-1b could heterodimerize with LBP-1a and LBP-1c (Yoon et al. 1994). Thus, we hypothesized that LBP-1b has the ability to transport LBP-1a and LBP-1c into the nucleus by forming heterodimers. In order to examine the possibility, we expressed YFP-LBP-1a or YFP-LBP-1c together with CFP-LBP-1b. As shown in Fig. 4, co-expression of CFP-LBP-1b resulted in the nuclear localization of YFP-LBP-1a and YFP-LBP-1c, although a weak signal from YFP-fusions was still observed in the cytosol. Co-expression of YFP-LBP-1c and CFP-LBP-1a did not change their cytosolic localization, and localization of YFP was unaffected by co-expression of CFP-LBP-1b.

View larger version (37K): [in this window] [in a new window]   Figure 4  Nuclear transport of LBP-1a and LBP-1c by LBP-1b. YFP-LBP-1 and CFP-LBP-1 chimeric plasmids were transfected into HeLa or COS-7 cells. Cells were fixed with 4% paraformaldehyde. Imaging was performed with an Olympus BX50 fluorescence microscope equipped with a filter set (Olympus U-MCFPHQ and U-MYFPHQ) for discriminating between YFP and CFP and the Olympus DP70 digital camera. Nuclei are labeled with DAPI. Scale bars, 20 µm.

The YFP-LBP-1b expressed in COS-7 cells formed speckles in the nucleus (Fig. 1), although the protein was diffused throughout the nucleus of HepG2 and HeLa cells. To investigate the subnuclear localization of endogenous LBP-1 proteins, COS-7 cells were immunostained with an antibody to NF2d9 (LBP-1a) (Fig. 5). Cross-reactivity of the antibody to LBP-1b and LBP-1c used for the immunostaining was confirmed by Western blotting. Expression level of the three proteins by DNA transfection in COS-7 cells was similar to each other (Fig. 5). Multiple bands were detected in each lane, and it may suggest a difference in the phosphorylation status of the proteins. Phosphorylation of LBP-1c by MAP kinases was reported (Volker et al. 1997). As shown in Fig. 5, LBP-1 proteins were observed in the form of speckles although the nucleoplasm was also immunoreactive to the antibody. No signal was found in the cytosol. In order to identify the nuclear speckles to which LBP-1 proteins were localized, we expressed YFP-LBP-1b in COS-7 cells and simultaneously immunostained the cells with antibodies to PML or coilin, marker proteins of the PML and Cajal bodies. Almost all of the speckles overlapped with the PML body as shown in Fig. 5. Cajal bodies stained with antibody to coilin did not overlap with the speckles. We expressed various deletion mutants of NLS-YFP-LBP-1a to investigate the region responsible for adhesion to the PML body in COS-7 cells as shown in Fig. 6. Analysis of deletion mutants by SDS-PAGE followed by immunoblotting using an antibody to YFP showed that deleted proteins were expressed and migrated to the positions with estimated molecular weights (data not shown). LBP-1a with C-terminal deletion to 414 formed speckles similar to full-length LBP-1a. Deletion to 277 still formed speckles, although the deleted protein also localized in the nucleoplasm. Further deletion of 15 amino acids (NLS-YFP-LBP-1a(1-261)) resulted in the elimination of the speckles. Since NLS-YFP-LBP-1a(1-276) formed speckles, we expressed a mutant, NLS-YFP-LBP-1a(41-273), with deletion of N-terminal 40 amino acids. The deleted protein still formed speckles. Further deletion of amino acids to 64 greatly reduced the ability to form speckles. NLS-YFP-LBP-1a(109-464) did not form speckles. Fragments covering central or C-terminal regions (NLS-YFP-LBP-1a(230-276), NLS-YFP-LBP-1a(379-464), and NLS-YFP-LBP-1a(277-504)) did not form speckles. Taken together, we conclude that the region comprising of amino acids 65-273 is necessary for accumulation into the PML body. This N-terminal region overlapped with the DNA binding domain of LBP-1 proteins (Zhong et al. 1994). Interestingly, a region that led to localization into round speckles differing from the PML body was found in the C-terminal half of LBP-1a (Fig. 6, NLS-YFP-LBP-1a(277-464) and NLS-YFP-LBP-1a(319-464)). These speckles may explain the imperfect overlap of speckles of LBP-1 with the PML body as observed in Fig. 5. Identification of the speckles remains to be clarified.

View larger version (20K): [in this window] [in a new window]   Figure 5  Accumulation of LBP-1 proteins into the PML body in COS-7 cells. Immunostaining of COS-7 cells was performed with an anti-LBP-1a antibody followed by the addition of a tetramethylrohdamine-conjugated second antibody. The cells were observed with an Olympus BX50 fluorescence microscope. A typical image of the nucleus is shown on the left. Cross-reactivity of the antibody to LBP-1b and LBP-1c used for the immunostaining was examined by Western blotting. Whole cell extracts from cells transfected with LBP-1 expression plasmids (lane 1, pBOSLBP-1a; lane 2, pBOSLBP-1b; lane 3, pBOSLBP-1c; lane 4, pEF-BOS) were subjected to Western blotting analysis for LBP-1 proteins as described under ‘Experimental procedures.’ A chimeric plasmid (pYFP-LBP-1b) was transfected into COS-7 cells and incubation was performed for 24 h. The cells were then immunostained with an anti-PML or coilin antibody followed by the addition of a tetramethylrohdamine-conjugated second antibody, and visualized by fluorescence microscopy with an Olympus BX50 fluorescence microscope.

View larger version (21K): [in this window] [in a new window]   Figure 6  Regions of LBP-1a necessary for adhesion to nuclear bodies. Regions of fragments in various deletion mutants are shown on the left. DNA fragments generated by PCR or restriction enzyme digestions of LBP-1a cDNA coding for 504 amino acids were inserted 3'-downstream of pNLS-YFP. These deletion plasmids were transfected into COS-7 cells. 24 h after transfection, the cells were fixed with 4% paraformaldehyde. Fluorescence of YFP chimeric proteins was detected with an Olympus BX50 fluorescence microscope. Nuclei are labeled with DAPI. Scale bars, 20 µm.

	Discussion

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In this study, we have clearly shown that LBP-1a and LBP-1c have no ability to enter into the nucleus per se and that LBP-1b transports the proteins into the nucleus. This finding implies that expression of LBP-1b is required for the nuclear function of LBP-1a and LBP-1c. We examined expression of the LBP-1b mRNA and compared it with that of the LBP-1a mRNA in several cultured cells by RT-PCR. Roughly equal amounts of expression were found between the two mRNAs in the cells examined (data not shown). The mechanism of nuclear localization of LBP-1d, which deletes a part of DNA binding region by alternative splicing, is presently not known. It has been shown that LBP-1d possesses an extremely low ability to interact with LBP-1c (Shirra et al. 1994). This finding suggests that LBP-1d may not be carried into the nucleus by LBP-1b, although interaction between LBP-1d and LBP-1b has not been examined. On this point, Zambrano et al. (1998) observed that LBP-1d is localized mainly in non-nuclear fractions. We do not exclude other mechanisms of nuclear localization of LBP-1a and LBP-1c. In fact, it is reported that LBP-1c forms complexes with Fe65 and the complex is present in both nuclear and non-nuclear fractions (Zambrano et al. 1998).

Recently, it has become widely recognized that the eukaryotic nucleus contains a number of distinct subcompartments that include the PML body and Cajal body. The PML body was first identified as autoantigens in primary biliary cirrhosis patients, and the PML gene was cloned as a chromosomal translocation partner of the retinoic acid receptor RAR in acute promyelocytic leukemia (APL) (Melnick & Licht 1999). Currently, biochemical and physiological functions of the PML body are not well understood. Defense systems to foreign and over-expressed proteins are postulated (Borden 2002). In addition to these functions, recent data indicate that a number of transcription factors and chromatin remodeling factors are associated with PML, strongly suggesting a transcriptional role of the PML body. Furthermore, it has been suggested that PML body represents a scaffold into which cofactor complexes are assembled to be utilized by transcription factors. In this respect, it is interesting to note that the corepressor Sin3A that has been shown to interact directly with LBP-1c (Drouin et al. 2002) could also interact with PML (Khan et al. 2001). Overlapping of necessary sequences for localization of LBP-1 proteins to the PML body with the DNA binding region may suggest hindrance of LBP-1 proteins to the binding to DNA when they reside on the PML body. The protein-protein interaction between LBP-1 proteins and Sin3A may accelerate on the PML body. In the regulation of LBP-1 proteins, it is important to elucidate the role of the PML body on which co-localization of LBP-1 proteins and Sin3A may occur.

In addition to this interaction, it has been shown that several proteins directly interact with LBP-1 proteins. Cooperation of LBP-1c with YY1 may selectively down-regulate HIV transcription (Romerio et al. 1997). It has been suggested that Fe65, a ligand of ß-amyloid precursor protein, could directly interact with LBP-1c and LBP-1d through its N-terminal PID/PTB (Zambrano et al. 1998). It is important to further investigate involvement of the PML body in the formation of these protein-protein interactions.

	Experimental procedures

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Construction of plasmids

LBP-1 expression plasmids containing full-length cDNA sequences, NF2d9/pCR3, NF2d9 L/pCR3 and CP2/pCR3 were kindly provided by Dr M. Negishi (NIEHS). NF2d9/pCR3, NF2d9 L/pCR3 and CP2/pCR3 were digested by AflII and XbaI for construction of pBOSLBP-1a, pBOSLBP-1b and pBOSLBP-1c. The resultant fragments were treated with Klenow fragment and inserted into the blunt-ended XbaI site of pEF-BOS (Mizushima & Nagata 1990). YFP-LBP-1a, YFP-LBP-1b and YFP-LBP-1c chimeric plasmids were constructed by inserting the Klenow-treated AflII-XbaI fragment into the blunt-ended EcoRI site of pEYFP-C1. The amino acid sequence of the linker region of the chimeric proteins is SGLRSRAQASNFK. CFP-LBP-1 chimeric plasmids were similarly constructed using pECFP-C1. LBP-1a-YFP and LBP-1b-YFP were constructed as follows. The stop codon of LBP-1a and LBP-1b was changed to CCG by PCR using primers, 5'-GCTACAACCCAGGAAACACA-3' and 5'-CGACCGGTTTCAAAATTATGTGAATACC-3'. After digestion with ApaI and Pin AI of the synthesized fragment, a 130 bp ApaI-PinAI fragment were inserted between the ApaI and Pin AI sites of pEYFP-NI to generate an intermediate plasmid. KpnI-ApaI fragments of NF2d9/pCR3 and NF2d9 L/pCR3 were inserted between the KpnI and ApaI sites of the intermediate plasmid to generate LBP-1a-YFP and LBP-1b-YFP. pLBP-1c-YFP was similarly constructed. The stop codon of LBP-1c was changed to CCG by PCR using primers, 5'-TCCTCACCAGATCAGCCAGA-3' and 5'-CGACCGGTTTGAGAATGACATGATAGCTG-3'. After digestion of the synthesized fragment by BamHI and Pin AI, a 120 bp BamHI-Pin AI fragment was inserted between BamHI and Pin AI site of pEYFP-NI to generate an intermediate plasmid. The 1.4 kb EcoRI-BamHI fragment of CP2/pCR3 was inserted between EcoRI and BamHI sites of the intermediate plasmid to generate LBP-1c-YFP. For construction of pNLS-YFP, NLS of LBP-1b was amplified by PCR using oligonucleotides, 5'-CCCGAATTCATACCACAATCCTCACAGAG-3' and 5'-CCCGGATCCGCATCGGGCCACGGAGAACA-3'. The amplified fragment was digested with EcoRI and BamHI, and inserted between the EcoRI and BamHI sites of pEYFP-N1. The inserted fragment encodes 36 amino acids arising from exon 6 and succeeding 7 amino acids The resultant plasmid was digested with AceI and BsrGI, and the fragment containing the NLS-YFP sequence was used to replace the AceI/BsrGI fragment of pEYFP-C1 to generate pNLS-YFP. Full-length LBP-1a cDNA, various fragments of LBP-1a cDNA were inserted into the 3' multicloning site of pNLS-YFP. pNLS-YFP-LBP-1a was constructed by inserting the KpnI/EcoRV fragment of YFP-LBP-1a between the KpnI and SmaI sites of NLS-YFP. For construction of pNLS-YFP-LBP-1a(1-413), the DNA fragment obtained by digestion of LBP-1a cDNA with KpnI and PstI was cloned between the KpnI and SmaI sites of pNLS-YFP after treating them with T4 polymerase. The same procedure was followed for the constructions as follows: for pNLS-YFP-LBP-1a(1-276), the fragment obtained with KpnI and NcoI was cloned between the KpnI and SmaI sites; for pNLS-YFP-LBP-1a(1-228), the fragment obtained with KpnI and PvuII was cloned between KpnI and SmaI sites; for pNLS-YFP-LBP-1a(277-464), the fragment obtained with NcoI and ApaI was cloned between KpnI and ApaI sites; for pNLS-YFP-LBP-1a(109-464), the fragment obtained with SacI and ApaI was cloned between the SalI and ApaI sites; for pNLS-YFP-LBP-1a(319-464), the fragment obtained with BstXI and ApaI was cloned between the KpnI and ApaI sites; for pNLS-YFP-LBP-1a(379-464), the fragment obtained with EcoRI and ApaI was cloned between the SalI and ApaI; and for pNLS-YFP-LBP-1a(277-504), the fragment obtained with NcoI and EcoRV was cloned into the KpnI site. pNLS-YFP-LBP-1a(1-261), pNLS-YFP-LBP-1a(230-276), pNLS-YFP-LBP-1a(41-273), and pNLS-YFP-LBP-1a(65-273) were constructed by inserting fragments, which were amplified by PCR followed by digestion with KpnI and ApaI, between the KpnI and ApaI sites of pNLS-YFP. Oligonucleotides used for PCR are as follows: 5'-CGGGGTACCATGGCCTGGGTGCTCAGTATG-3' and 5'-ATGGGGGCCCCTTTTCTTTTTCATGAGCTGT for pNLS-YFP-LBP-1a(1-261); 5'-CGGGGTACCTGCCAAATCAAAGTCTTTAAG-3' and 5'-ATGGGGGCCCTGGAGAACACTCTGTGAGAAT-3' for pNLS-YFP-LBP-1a(230-276); 5'-GGTACCTTGGCATTGCCCATTTTCAAGCAAGAAGATTCC-3' and 5'-GGGCCCCTCTGTGAGAATGGTGGTGTCGTAAGATGG-3' for pNLS-YFP-LBP-1a(41-273); 5'-GGTACCCAATATGTAATGTGTGCTGCAACTTCGCCAGC-3' and 5'-GGGCCCCTCTGTGAGAATGGTGGTGTCGTAAGATGG-3' for pNLS-YFP-LBP-1a(65-273). Mutant plasmids, pKK16,17AA, pKR20,21AA and pKR33,34AA, with mutations in the NLS were constructed using the QuickChange site-directed mutagenesis kit (Stratagene). All DNA constructions were validated by sequence analysis.

Western blotting

Whole cell extracts were prepared from COS-7 cells transfected with pBOSLBP-1a, pBOSLBP-1b or pBOSLBP-1c by mixing 10 mM HEPES buffer, pH 7.9, containing 0.1 mM EDTA, 0.4 M NaCl, 0.5% NP-40, 0.2 mM sodium orthovanadate, 50 mM sodium fluoride, 10 µg/mL aporotinin and 1 mM phenylmethylsulfonyl fluoride. Proteins were resolved by 8.0% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Biosciences). A rabbit anti-NF2d9 (LBP-1a) antibody (a generous gift from Dr M. Negishi) diluted 1 : 500 was used as the first antibody. Goat anti-rabbit biotinylated IgG (Vector Laboratories) was used as the second antibody. The membrane was developed by the ECL (Amersham Biosciences).

Fluorescence observation of cells

Human hepatoma HepG2 cells were maintained in MEM supplemented with 10% fetal bovine serum, 1% nonessential amino acid solution (Invitrogen) and 0.1% sodium pyruvate (Invitrogen). HeLa and COS-7 cells were grown in MEM and DMEM, respectively, supplemented with 10% fetal bovine serum. Cells grown on the cover glass were transfected with 0.3 µg YFP-fusion plasmids using FuGENE6 transfection reagent (Roche), and incubated for 24 h (COS-7 cells) or 48 h (HepG2 and HeLa cells) before fixation in 4% paraformaldehyde in PBS, pH 7.4. The fixed cells were incubated in 0.2 µg/mL of DAPI (4’,6’-diamidino-2-phenylindole) labeling solution at room temperature for 5 min. Imaging was performed with an Olympus BX50 fluorescence microscope equipped with a filter set (Olympus U-MCFPHQ and U-MYFPHQ) for discriminating between YFP and CFP and the Olympus DP70 digital camera. The fluorescent color of DAPI was changed from blue to red on a computer.

Immunofluorescent staining of COS-7 cells

COS-7 cells grown on the cover glass were fixed in 4% paraformaldehyde in PBS, pH 7.4, for 15 min at room temperature. The fixed cells were washed three times in PBS and then permeabilized for 5 min in PBS containing 0.5% Triton X-100. Cells were washed and incubated with 1 : 500 diluted anti-NF2d9 (LBP-1a) antibody or anti-PML (1: 500 diluted) (Santa Cruz Biotechnology) and anti-p80 coilin (1: 500 diluted) (BD Transduction Laboratories) antibodies for 1 h at room temperature. The cells were washed in PBS and then incubated in tetramethylrhodamine-conjugated secondary antibody at a dilution of 4 µg/mL for 1 h. The immunostained cells were washed 4 times in PBS, and imaging was performed with an Olympus BX50 fluorescence microscope equipped with an Olympus DP70 digital camera.

	Acknowledgements

 
We thank Dr M. Negishi (NIEHS) for the generous gift of LBP-1 cDNAs and an antibody to NF2d9. This work was supported in part by Grant-in-Aid for research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Footnotes

 
Communicated by: Hiroshi Hamada

^aPresent address: Department of Cell Biology, Graduate School of Medicine and School of Medicine, Tohoku University, Sendai 980-8575, Japan

^* Correspondence: E-mail: sogawa{at}mail.tains.tohoku.ac.jp

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Received: 16 March 2005
Accepted: 22 May 2005

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