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1 Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
2 Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8577, Japan
3 School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia
4 University of Queensland Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, Ipswich Road, Brisbane, Qld 4102, Australia
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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XX (: hydrophobic amino acids) motifs, within the negative regulatory domain of c-Myb. The XX motif was initially demonstrated to be critical for interaction between nuclear hormone receptors and their co-activators (Heery et al. 1997). In addition to Mybbp1a, multiple co-repressors, including TIF1β and BS69, also bind to the leucine-rich region of c-Myb, and suppress c-Myb activity (Ladendorff et al. 2001; Nomura et al. 2004). Point mutations in this leucine-rich region enhance the c-Myb's capacity to activate transcription and to transform hematopoietic cells (Kanei-Ishii et al. 1992). Although p67MBP reduces the trans-activating capacity of c-Myb by 80%, p160MBP does not affect c-Myb activity (Tavner et al. 1998). What kinds of signals can induce the processing of p160MBP into p67MBP remains unknown, and why these two forms have different capacities to regulate c-Myb activity?
Mybbp1a also binds to a number of other transcription factors. Both p160MBP and p67MBP bind to the transcriptional co-activator PPAR
co-activator 1
(PGC-1) (Fan et al. 2004), which is a key regulator of metabolic processes (Lin et al. 2005). Binding of either form of Mybbp1a to PGC-1 represses the co-activator activity of PGC-1, while the interaction between PGC-1 and Mybbp1a is disrupted by p38 MAPK phosphorylation of PGC-1. p160MBP also directly binds to the transcription activation domain of the RelA/p65 subunit of NF-
B, and suppresses its trans-activating capacity by competing with p300 for binding to the trans-activation domain of RelA/p65 (Owen et al. 2007). p160MBP was recently identified as an component of the co-repressor Ret–CoR complex, which mediates photoreceptor cell-specific nuclear receptor (PNR)-dependent repression (Takezawa et al. 2007), although the role of p160MBP in this complex is unknown. These findings suggest that Mybbp1a functions as a co-repressor in many situations, although Mybbp1a can also bind to the aryl hydrocarbon receptor (AhR), a basic helix–loop–helix/Per–Arnt–Sim (bHLH/PAS) transcription factor, to stimulate the transcriptional activation capacity of AhR (Jones et al. 2002).
Endogenous Mybbp1a is predominantly localized in the nucleolus, although nucleoplasmic localization is also observed (Tavner et al. 1998). Deletion analysis of p160MBP indicated that its carboxyl terminus, which contains several short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization (Keough et al. 2003). On the other hand, p67MBP is mainly localized in the cytoplasm when it is over-expressed by itself, suggesting that endogenous p67MBP is localized in the nuclei via interaction with another protein(s). Although the role of Mybbp1a in the nucleolus is unknown, Mybbp1a has some homology with Po15p of yeast Saccharomyces cerevisiae, which is required for rRNA production (Shimizu et al. 2002).
Here, we report that the stress signals which inhibit ribosomal biosynthesis induce the processing of p160MBP into p140MBP and p67MBP, and their translocation from the nucleolus to the nucleoplasm.
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Results |
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A HeLa S3 cell lines in which p160MBP tagged with both FLAG and HA is stably expressed, were generated by retroviral transduction. By Western blotting analysis, we selected one of several clones that expressed FLAG/HA-p160MBP at the lowest level, and used this clone to purify the p160MBP complex, because its high level expression may induce imbalance between the components of the complex. Cells were disrupted in hypotonic buffer, and nuclei isolated. The p160MBP complex was purified from nuclear extracts by sequential immunoprecipitation with anti-FLAG and anti-HA antibodies, as described by Nakatani & Ogryzko (2003), followed by glycerol gradient centrifugation. Silver staining of SDS-PAGE gels of the purified proteins revealed two different-sized complexes (Fig. 1A). Mass spectrometric analysis indicated that the small complex contained p140MBP, which lacks the C-terminal region of p160MBP as shown below, nucleolin (also called C23), p67MBP, which is a an N-terminal fragment of Mybbp1a generated from p160MBP, EBP1 (Erb3-binding protein) and nucleophosmin (also called B23). The large complex involved p160MBP, p140MBP, topoisomerase I, nucleolin, nucleostemin, nucleophosmin, histone H1x and many ribosomal proteins. Nucleolin, nucleostemin and nucleophosmin are nucleolar proteins, but are also localized in the nucleoplasm to some extent. Nucleolin is an acidic phosphoprotein and is involved in the transcriptional control of ribosomal RNA (rRNA) genes, in ribosome maturation and assembly, and in nucleocytoplasmic transportation of ribosomal components (Mongelard & Bouvet 2007). Nucleostemin is a GTP-binding protein highly enriched in the stem cells and cancer cells (Tsai & McKay 2002). Nucleophosmin is a phosphoprotein that plays roles in multiple steps of ribosome biogenesis, including ribosome assembly and transport, and is frequently over-expressed, mutated and deleted in cancers (Grisendi et al. 2006). EBP1 (also called PA2G4: proliferation-associated 2G4) was originally isolated using the antibody mAb2G4 which recognized the single-strand DNA-binding protein, and is highly expressed between G1 and mid-S phase (Radomski & Jost 1995).
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Both p140MBP and p67MBP were detected in the small Mybbp1a complex, while the large complex contained both p160MBP and small amount of p140MBP. These results raised the possibility that Mybbbp1a may form a dimer or an oligomer. To examine this, we investigated whether the p160MBP proteins intermolecularly interact each other using the GST-pull-down assays. In vitro-translated p160MBP bound to the GST fusion proteins containing p67MBP or the C-terminal 299-amino acids fragment of p160MBP, while it did not interact with the GST protein alone (Fig. 1D). These results suggest that the Mybbp1a molecules can form a dimer or an oligomer.
Generation of p140MBP by removal of the C-terminal basic region of p160MBP
The difference in the molecular weight between p160MBP and p p140MBP was about 20–40 kDa, which was not likely due to modification such as phosphorylation. We examined the peptides generated from p140MBP, which were identified by mass spectrometric analysis. The peptides detected covered the region between amino acids 6 and 1170 of p160MBP but no peptides were found that were derived from the C-terminal 174-amino acids of p160MBP (Fig. 2), suggesting that p140MBP is generated by removal of the C-terminal 170-amino acids. This C-terminal region contains a cluster of basic amino acids similar to a cluster in the N-terminal region of ARF that was reported to be responsible for nucleolar localization (Weber et al. 1999). Furthermore, we (RAK and TJG) previously demonstrated that the C-terminal 193 amino acids of p160MBP was sufficient to confer nuclear and nucleolar localization on β-galactosidase protein (Keough et al. 2003). Together with these reports, our results suggest that the small complex containing p140MBP and p67MBP is generated by processing and may translocate from the nucleolus to the nucleoplasm.
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A recent report on mass-spectrometric analysis of nucleolar proteins (Andersen et al. 2005) indicated that Mybbp1a is one such protein that is depleted from the nucleolus by treatment of cells with actinomycin D (ActD), which selectively inhibits rRNA gene transcription by low doses (Sollner-Webb & Tower 1986; Scheer & Weisenberger 1994). Very recently, Mybbp1a was shown to translocate from the nucleolus to the nucleoplasm by ActD treatment (Diaz et al. 2007). These results suggested the possibility that ActD treatment induces the processing of p160MBP to p140MBP and p67MBP, and subsequent translocation of the small complex containing p140MBP and p67MBP from the nucleolus to the nucleoplasm. To examine this, we treated HeLa cells expressing FLAG/HA-p160MBP with ActD, and prepared cell lysates at 12 h after ActD addition, which were then used for immunoprecipitation with anti-FLAG antibody followed by Western blotting with anti-Mybbp1a antibody. ActD treatment reduced the levels of p160MBP by about 50%, and increased the levels of p140MBP and p67MBP (Fig. 3A), suggesting that ActD treatment induces the processing of p160MBP to p140MBP and p67MBP.
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ActD induces the translocation of the p67MBP/p140MBP complex from the nucleolus to the nucleoplasm
To examine whether ActD induces a translocation of p140MBP and p67MBP from the nucleolus to the nucleoplasm, HeLa cells expressing FLAG/HA-p160MBP were treated with ActD, and the subcellular localization of FLAG/HA-Mybbp1a was analyzed. The FLAG/HA-Mybbp1a signals detected with the anti-FLAG antibody were predominantly in the nucleolus in the ActD-untreated cells (Fig. 3B). When cells were treated with ActD, significant amounts of Mybbp1a signals were detected in the nucleoplasm. The degree of increase in the levels of nucleoplasmic FLAG/HA-Mybbp1a by ActD treatment appears to be consistent with the degree of processing of p160MBP to p140MBP and p67MBP shown in Fig. 3A.
To confirm the translocation of Mybbp1a, we utilized time-lapse microscopic analysis. For this purpose, we generated HeLa cells expressing a fusion of Venus, a GFP derivative (Nagai et al. 2002) with p160MBP (Venus-p160MBP) (Fig. 4A). Time-lapse analysis of the subcellular localization of Venus-p160MBP indicated that significant amounts of the Venus-Mybbp1a signal translocated from the nucleolus to the nucleoplasm from 30 min after ActD addition and reached plateau at about 2 h (Fig. 4B). When ActD was washed out, Venus-Mybbp1 in the nucleoplasm was not re-translocated to the nucleoli at least during 6 h after ActD removal (Fig. 4C), although nucleophosmin re-translocated to the nucleoli within 6 h after ActD removal (data not shown). These results suggest that translocation of Mybbp1a to the nucleoplasm from the nucleoli is irreversible.
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To examine whether ActD induces a translocation of endogenous Mybbp1a from the nucleolus to the nucleoplasm, NIH3T3 cells were treated with ActD, and the subcellular localization of endogenous Mybbp1a was analyzed. The signals detected with the antibody raised against the N-terminal region of p160MBP, which include p160MBP, p140MBP and p67MBP, were predominantly in the nucleolus in the ActD-untreated cells (Fig. 6). When cells were treated with ActD, significant amounts of Mybbp1a signals were detected in the nucleoplasm. After ActD treatment, nucleophosmin, which was involved in both small and large complexes, were also translocated from the nucleolus to some extent. Treatment of NIH3T3 cells with cisplatin or UV induced a translocation of Mybbp1a signals from the nucleolus into the nucleoplasm (Fig. 6). Nucleophosmin was also similarly translocated from the nucleolus to the nucleoplasm by treatment with cisplatin or UV irradiation.
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Nucleophosmin was detected in both small and large Mybbp1a complexes (Fig. 1A,B). Nucleophosmin was recently shown to be a component of a complex containing CTCF (CCCTC-binding factor), which binds to insulator elements and blocks enhancers of one gene from activating a promoter on another nearby gene (Yusufzai et al. 2004). The CTCF complex also contained topoisomerase II, histone H1x and several 40S ribosomal subunit proteins, while the large Mybbp1a complex contained topoisomerase, histone H1x and many ribosomal subunits. This similarity raised the possibility that the Mybbp1a complexes may interact with CTCF, which we examined using co-immunoprecipitation. Cell lysates from HeLa cells expressing FLAG/HA-p160MBP were immunoprecipitated with anti-FLAG antibody, and the immunocomplexes were subjected to Western blotting with anti-CTCF antibody. Figure 7A shows that CTCF was co-immunoprecipitated with FLAG/HA-p160MBP and furthermore, that poly(ADP-ribose) polymerase (PARP), which has been detected in the CTCF complex (Yusufzai et al. 2004), was also co-immunoprecipitated with FLAG/HA-p160MBP.
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Discussion |
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The large Mybbp1a complex contained nucleolin, nucleostemin, nucleophosmin and many ribosomal subunit proteins. Nucleolin and nucleophosmin are known to stimulate ribosome biogenesis by participating in multiple steps, including the transcription of rRNA genes, ribosome maturation, assembly and ribosome transport (Grisendi et al. 2006; Mongelard & Bouvet 2007). The fact that nucleostemin, a GTP-binding protein, is enriched in the actively proliferating cells, such as stem cells and cancer cells (Tsai & McKay 2002), is also consistent with its possible role in ribosome biogenesis. Thus, the role of these three nucleolar proteins and the involvement of many ribosomal subunits strongly suggest that the large Mybbp1a complex stimulates ribosome biogenesis. Consistent with this, Mybbp1a has a significant homology with the yeast protein Pol5p, which were shown to bind to the rRNA gene promoter (Shimizu et al. 2002; Nadeem et al. 2006). Although Pol5p shows some similarity with DNA polymerases, subsequent computational analysis of the sequence indicated that Pol5p is not a DNA polymerase (Yang et al. 2003).
The small Mybbp1a complex contained nucleolin and nucleophosmin in addition to p140MBP and p67MBP. As both p140MBP and p67MBP lack the nucleolar localization signals in the C-terminal region of p160MBP, this complex is likely to be translocated into the nucleoplasm immediately after processing where it can interact with various transcription factors. Previously, we demonstrated that p67MBP, but not p160MBP, inhibited the c-Myb-dependent trans-activation (Tavner et al. 1998). This may be because over-expression of p160MBP does not lead to an increase in the amounts of the small Mybbp1a complex in the nucleoplasm. Although the mechanism by which the small Mybbp1a complex inhibits the c-Myb activity remains unknown, it was reported that nucleolin directly binds to the DNA-binding domain of c-Myb and inhibits the c-Myb-dependent trans-activation (Ying et al. 2000).
The Mybbp1a complexes and the CTCF complex share some components: nucleophosmin, topoisomerase, some ribosomal subunits and histone H1x. Furthermore, CTCF was co-immunoprecipitated with Mybbp1a, and detected in the purified Mybbp1a complexes. Yusufzai et al. (2004) previously reported the evidence that the insulator, which binds to CTCF, is drawn to the nucleolar surface by its strong interaction with nucleophosmin, and proposed that the CTCF–nucleophosmin complex creates separate loop domain structures to prevent the passage of a processive activating complex from the distal enhancer to the promoter. Therefore, the small Mybbp1a complex may also suppress transcription from Myb binding site-containing promoters by recruiting Myb bound to such sites to the nucleolar surface. We have detected CTCF not only in the small Mybbp1a complex but also in the large Mybbp1a complex, which appears to be in the nucleolus. Recently, it was reported that CTCF is localized in the nucleoli of differentiated cells and nucleolar localization of CTCF is associated with growth arrest and inhibition of rRNA gene transcription (Torrano et al. 2006). Thus, CTCF may inhibit ribosome biogenesis by interacting with the Mybbp1a large complex in the nucleoli.
As transcription of rRNA genes and the biosynthesis of ribosomes in the nucleolus are the major and limiting metabolic activities, the rate of ribosome biogenesis is tightly linked to cellular proliferation. Furthermore, recent data suggest that the nucleolus also plays an important role in cell-cycle regulation and senescence (Guarente 1997; Sherr & Weber 2000; Visintin & Amon 2000). ActD, cisplatin and UV irradiation, all of which inhibit ribosome biogenesis, induced the translocation of the small Mybbp1a complex from the nucleolus to the nucleoplasm. We suggest that the nucleolar large Mybbp1a complex acts to stimulate the ribosome biogenesis, while the nucleoplasmic small Mybbp1a complex may bind to c-Myb, leading to suppression of c-Myb-dependent transcription. c-Myb positively regulates cell cycle progression by activating several cell-cycle regulating genes, such as c-myc and cyclin B1 (Nakagoshi et al. 1992; Nakata et al. 2007). c-Myb also suppresses apoptosis by inducing some anti-apoptotic genes, such as bcl-2 (Frampton et al. 1996; Taylor et al. 1996). Thus, translocation of Mybbp1a from the nucleolus to the nucleoplasm upon ribosome stress may play an important role to block the cell cycle progression and to induce apoptosis by suppressing these c-Myb target genes. Mybbp1a also binds to the RelA/p65 subunit of NF-B and PGC-1. Like c-Myb, NF-B positively regulates cellular proliferation (Viatour et al. 2005), while PGC-1 is a key regulator for energy production in mitochondria (Lin et al. 2005). Therefore, the inhibition of NF-B and PGC-1 by Mybbp1a may also contribute to suppression of cellular proliferation and energy production upon ribosome stress. Thus, Mybbp1a could be a key regulator that connects ribosome biogenesis and transcription to control cell cycle progression, proliferation, and energy production, as illustrated in Fig. 8.
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Experimental procedures |
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To generate a retroviral vector expressing FLAG- and HA-tagged p160MBP, the mouse p160MBP cDNA was cloned into the pOZ-FH-N vector (Nakatani & Ogryzko 2003). The vector was transfected into the amphotropic packaging Phoenix A cell line and medium containing the amphotropic virus was prepared. HeLa S3 cells were transduced with a recombinant retrovirus expressing a bicistronic mRNA encoding FLAG/HA-p160MBP linked to the IL-2 receptor subunit, and the transduced subpopulation was purified by repeated cycles of affinity cell sorting. After sorting, cells were plated onto 96-well plate and single cell clones were isolated. Cells were disrupted in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT), and the nuclear pellet was collected by centrifugation at 25 000 g for 20 min. The pellet was extracted with buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) for 30 min at 4 ºC and lysates were collected by centrifugation at 25 000g for 30 min. The p160 complex was immunoprecipitated from nuclear extracts prepared from HeLa cells expressing p160 by incubating with M2 anti-FLAG agarose (Sigma, St. Louis, MO) for 4 h with rotation. After an extensive wash with wash buffer (20 mM Tris–HCl, pH 8.0, 0.1 M KCl, 5 mM MgCl2, 10% glycerol, 1 mM PMSF, 0.1% Tween 20, 10 mM β-mercaptoethanol), the bound proteins were eluted from the M2 agarose by incubation for 30 min with 0.2 mg/mL FLAG peptide (Sigma) in the same buffer. The eluates were further purified by immunoprecipitation with protein G-Sepharose (Amersham, Uppsala, Sweden) conjugated to the anti-HA 12CA5 antibody. The bound proteins were eluted from the matrix by incubating for 60 min with 0.5 mg/mL HA peptide in wash buffer. The purified proteins was separated by 4%–20% gradient SDS polyacrylamide gel electrophoresis (SDS-PAGE) and silver stained. For glycerol gradient sedimentation, 200 µL of FLAG antibody-immunoprecipitated material was loaded onto a 4.2-mL 10%–40% glycerol gradient in wash buffer. After centrifugation at 55 000 rpm for 1 h (Beckman, SW55Ti), 200 µL fractions were collected from the top of the gradient and resolved by 4%–20% gradient SDS-PAGE and silver stained. The p160 complex fractions were TCA precipitated, resolved by 4%–20% gradient SDS-PAGE and stained with Coomassie Blue. Protein bands were excised and analyzed by a Biflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA).
Culture and transfection of cells
HeLa cells and NIH3T3 cells were cultured in MEM or DMEM supplemented with 10% calf serum (CS), 100 U/mL penicillin G sodium and 100 µg/mL streptomycin sulfate at 37 ºC and in 5% CO2.
Co-immunoprecipitation
HeLa S3 cells expressing FLAG/HA-p160MBP from a 200 mL culture were disrupted in hyptonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT), and the nuclear pellet was collected by centrifugation at 15 000 g for 20 min. The pellet was extracted with buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) for 30 min at 4 ºC and lysates were collected by centrifugation at 15 000 g for 30 min. The Mybbp1a complex was immunoprecipitated from the extracts by incubating with M2 anti-FLAG agarose (Sigma) for 4 h with rotation. After five washes with wash buffer (20 mM Tris–HCl, pH 8.0, 0.1 M KCl, 5 mM MgCl2, 10% glycerol, 1 mM PMSF, 0.1% Tween 20, 10 mM β-mercaptoethanol), the bound proteins were eluted from M2 agarose by incubation for 30 min with 0.2 mg/mL FLAG peptide (Sigma) in the same buffer. The immunocomplexes were analyzed by SDS-PAGE, followed by Western blotting using anti-Mybbp1a (Tavner et al. 1998); anti-EBP1 (07–397, Upstate, Billerica, MA); anti-nucleophosmin (18–7288, Zymed, San Francisco, CA); anti-C23 (nucleolin) (sc-8031, Santa Cruz Biotechnology, Santa Cruz, CA) anti-nucleostemin (AF1638, R&D Systems, Minneapolis, MN); anti-CTCF (06–917, Upstate); anti-Histone H1 (05–457, Upstate); and anti-PARP (SA-253, Biomol, Plymouth Meeting, PA). Anti-Mybbp1a antibodies used a mixture of two polyclonal antibodies raised against the N-terminal 326 residues or C-terminal 298 residues of p160MBP (Tavner et al. 1998).
GST pull-down assay
GST pull-down assay was performed as described (Nomura et al. 1999). The binding buffer used consisted of 20 mM HEPES, pH 7.9, 75 mM KCl, 0.05% NP-40, 1.25 mM MgCl2, 0.1 mM EDTA, 1 mM DTT. GST fusion proteins bound to Glutathione-Sepharose 4B were mixed with 35S-p160 translated in vitro, and the bound proteins were analyzed by SDS-PAGE, followed by autoradiography.
Generation of HeLa cells expressing Venus-p160MBP
A retrovirus construct expressing a p160MBP fusion protein with Venus, a GFP derivative (Nagai et al. 2002), was constructed using the pOZ-FH-N vector after removal of the FLAG-HA tag, and virus was prepared as described above. HeLa cells expressing Venus-p160MBP were selected as described above. To detect the Venus-p160MBP proteins, cell lysates were prepared using buffer C, and subjected to SDS-PAGE, followed by Western blotting with anti-GFP (598, Medical Biological Laboratories, Nagoya, Japan).
Immunofluorescence microscopy
Cells were grown in 35 mm Petri dishes, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. After incubation for 60 min with 3% skim milk in PBS, cells were incubated for 1 h with the following primary antibodies: anti-Mybbp1a (rabbit polyclonal); anti-FLAG (M2, Sigma); anti-EBP1 (07–397, Upstate); anti-nucleophosmin (18–7288, Zymed); anti-C23 (nucleolin) (sc-8031, Santa Cruz Biotechnology); and anti-nucleostemin (AF1638, R&D Systems). Anti-Mybbp1a antibody used here was raised against the N-terminal 326 residues of p160MBP, and recognize p160MBP, p140MBP and p67MBP. The cells were washed, incubated with Alexa Fluor 488- or Cy3- or Rhodamine-conjugated anti-rabbit, anti-mouse or anti-goat secondary antibodies (Molecular Probes, Eugene, OR or Chemicon,Temecula, CA). Chromatin was labeled with TOTO-3 iodide (Molecular Probes).
Confocal images were obtained using an LSM510 (Zeiss, Jena, Germany) laser scanning microscope. In order to minimize overlapping signals, images were obtained by sequential excitation at 488/543/633 nm, to detect Alexa Fluor 488, Cy3 and TOTO-3, respectively, and emission signals were detected at 505–530 nm for Alexa Fluor 488, > 560 nm for Cy3 and > 650 nm for TOTO-3 iodide. Images were processed using Photoshop software.
Time-lapse imaging
Cells were cultured in poly-D-lysine coated glass bottom 35 mm dishes (IWAKI, Tokyo, Japan). For observation of living cells, medium was replaced with fresh DMEM containing 20 mM HEPES (pH 7.4). Dishes were imaged at 37 ºC using a Tempcontrol 37-2 chamber (Zeiss). Images were obtained using an LSM510 (Zeiss) laser scanning microscope. Images were captured at set time intervals for a period of at least 3 h. Images were processed using the Zeiss LSM image software.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: sishii{at}rtc.riken.jp
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References |
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Received: 13 August 2007
Accepted: 1 October 2007
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