Nuclear Localization and Cell Cycle-specific Expression of CtIP, a Protein That Associates with the BRCA1 Tumor Suppressor
2000; Elsevier BV; Volume: 275; Issue: 24 Linguagem: Inglês
10.1074/jbc.m909494199
ISSN1083-351X
Autores Tópico(s)Chromosomal and Genetic Variations
ResumoThe BRCA1 tumor suppressor has been implicated in a diverse spectrum of cellular processes, including transcriptional regulation, DNA repair, and cell cycle checkpoint control. CtIP was recently identified as a protein that associates with BRCA1 and two other nuclear factors, CtBP1 and Rb1. To understand the functions of CtIP, we have evaluated its biological properties with respect to those of BRCA1. Our results show that CtIP, like its associated factors, is predominantly a nuclear protein. A subset of the endogenous pool of CtIP polypeptides exists in a protein complex that includes both BRCA1 and the BRCA1-associated RING domain protein (BARD1). At the protein level, CtIP expression varies with cell cycle progression in a pattern identical to that of BRCA1. Thus, the steady-state levels of CtIP polypeptides, which remain low in resting cells and G1 cycling cells, increase dramatically as dividing cells traverse the G1/S boundary. In contrast to BRCA1, however, the G1/S induction of CtIP expression is mediated primarily by post-transcriptional mechanisms. Finally, the interaction between CtIP and BRCA1 is shown to be stable in the face of genotoxic stress elicited by treatment with UV light, adriamycin, or hydrogen peroxide. Together, these results indicate that CtIP can potentially modulate the functions ascribed to BRCA1 in transcriptional regulation, DNA repair, and/or cell cycle checkpoint control. The BRCA1 tumor suppressor has been implicated in a diverse spectrum of cellular processes, including transcriptional regulation, DNA repair, and cell cycle checkpoint control. CtIP was recently identified as a protein that associates with BRCA1 and two other nuclear factors, CtBP1 and Rb1. To understand the functions of CtIP, we have evaluated its biological properties with respect to those of BRCA1. Our results show that CtIP, like its associated factors, is predominantly a nuclear protein. A subset of the endogenous pool of CtIP polypeptides exists in a protein complex that includes both BRCA1 and the BRCA1-associated RING domain protein (BARD1). At the protein level, CtIP expression varies with cell cycle progression in a pattern identical to that of BRCA1. Thus, the steady-state levels of CtIP polypeptides, which remain low in resting cells and G1 cycling cells, increase dramatically as dividing cells traverse the G1/S boundary. In contrast to BRCA1, however, the G1/S induction of CtIP expression is mediated primarily by post-transcriptional mechanisms. Finally, the interaction between CtIP and BRCA1 is shown to be stable in the face of genotoxic stress elicited by treatment with UV light, adriamycin, or hydrogen peroxide. Together, these results indicate that CtIP can potentially modulate the functions ascribed to BRCA1 in transcriptional regulation, DNA repair, and/or cell cycle checkpoint control. glutathioneS-transferase polyacrylamide gel electrophoresis The BRCA1 tumor suppressor gene encodes a large polypeptide of 1863 amino acids that contains at least two recognizable protein motifs: a RING domain at the N terminus (1.Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Crossref PubMed Scopus (5330) Google Scholar) and two tandem BRCT repeats at the C terminus (2.Koonin E.V. Altschul S.F. Bork P. Nat. Genet. 1996; 13: 266-267Crossref PubMed Scopus (361) Google Scholar). To date, more than 200 different germline mutations of BRCA1 have been implicated in familial breast cancer (reviewed in Ref. 3.Couch F.J. Weber B.L. The Breast Cancer Information CoreHum. Mutat. 1996; 8: 8-18Crossref PubMed Scopus (270) Google Scholar). Although most of these are frameshift or nonsense mutations that grossly truncate the BRCA1 reading frame, in some cases breast cancer susceptibility has been attributed to more subtle defects that affect the BRCT coding sequences. These include missense mutations that cause single amino acid substitutions in either the first (A1708E) or second (M1775R) BRCT domain and a nonsense mutation that truncates the second BRCT domain by deleting 11 residues from the C terminus of BRCA1 (Y1853Δ). The fact that these mutations confer susceptibility to breast cancer implies that the BRCT domains play a crucial role in BRCA1-mediated tumor suppression. Although its molecular functions remain obscure, recent studies have implicated BRCA1 in several cellular processes, including cell growth control, transcriptional regulation, and the maintenance of genomic stability (reviewed in Ref. 4.Zhang H. Tombline G. Weber B.L. Cell. 1998; 92: 433-436Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The expression of BRCA1 varies with cell cycle progression in most established cell lines (5.Chen Y. Farmer A.A. Chen C.-F. Jones D.C. Chen P.-L. Lee W.-H. Cancer Res. 1996; 56: 3168-3172PubMed Google Scholar, 6.Gudas J.M. Li T. Nguyen H. Jensen D. Rauscher III, F.J. Cowen K.H. Cell Growth Differ. 1996; 7: 717-723PubMed Google Scholar, 7.Vaughn J.P. Davis P.L. Jarboe M.D. Huper G. Evans A.C. Wiseman R.W. Futreal P.A. Marks J.R. 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A. 1997; 94: 5820-5825Crossref PubMed Scopus (98) Google Scholar). Interestingly, this growth inhibition is abolished by tumor-associated lesions in the BRCT domains of BRCA1, including the A1708E and M1775R missense mutations. It has also been reported that overexpression of wild type BRCA1 inhibits S phase progression (15.Somasundaram K. Zhang H. Zeng X.Y. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (472) Google Scholar) and that a C-terminal segment of BRCA1 (residues 1293–1863) can ablate the G2/M checkpoint of human mammary epithelial cells, perhaps by dominant-negative inhibition of endogenous BRCA1 (16.Larson J.S. Tonkinson J.L. Lai M.T. Cancer Res. 1997; 57: 3351-3355PubMed Google Scholar). Although sequence-specific DNA recognition by BRCA1 has not been observed, experiments with GAL4p fusion proteins have shown that the C-terminal sequences of BRCA1 affect RNA transcription both in vivo and in vitro. Two groups have reported that a hybrid polypeptide containing the DNA binding domain of GAL4p (residues 1–147) fused to the BRCT sequences of BRCA1 (residues 1528–1863) can activate the transcription of GAL4p-responsive reporter genes and that this transactivation potential is ablated by tumor-associated mutations such as A1708E, M1775R, and Y1853Δ (17.Chapman M.S. Verma I.M. Nature. 1996; 382: 678-679Crossref PubMed Scopus (437) Google Scholar, 18.Monteiro A.N.A. August A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13595-13599Crossref PubMed Scopus (428) Google Scholar). Recently, a similar fusion protein that includes GAL4p residues 1–147 and BRCA1 residues 1560–1863 was shown to activate gene transcription in vitro(19.Haile D.T. Parvin J.D. J. Biol. Chem. 1999; 274: 2113-2117Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and alter chromatin structure in vivo (20.Hu Y.F. Hao Z.L. Li R. Genes Dev. 1999; 13: 637-642Crossref PubMed Scopus (107) Google Scholar). It has also been reported that BRCA1 co-purifies with the RNA polymerase II holoenzyme complex (21.Scully R. Anderson S.F. Chao D.M. Wei W. Ye L. Young R.A. Livingston D.M. Parvin J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5605-5610Crossref PubMed Scopus (425) Google Scholar, 22.Anderson S.F. Schlegel B.P. Nakajima T. Wolpin E.S. Parvin J.D. Nat. Genet. 1998; 19: 254-256Crossref PubMed Scopus (342) Google Scholar). These results as well as other data (15.Somasundaram K. Zhang H. Zeng X.Y. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (472) Google Scholar,23.Ouchi T. Monteiro A.N.A. August A. Aaronson S.A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. 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The HBL-100, T24, and HCT116 cell lines were obtained from the American Type Tissue Culture Collection. To generate CtIP-specific antibodies, glutathioneS-transferase (GST)1 fusion proteins that contain different segments of human CtIP were produced inEscherichia coli. Each fusion protein was then purified as described below and used to immunize rabbits or mice. The 14-1 mouse monoclonal antibody was raised against a GST fusion protein containing the C-terminal 278 amino acids of CtIP (residues 620–897). Rabbit polyclonal antisera 210 and 211 were raised against a GST fusion containing the C-terminal 208 amino acids of CtIP (residues 690–897). Rabbit antisera 164 and 614 were generated by immunizing with a GST fusion containing CtIP residues 58–369. The CtIP/pSP6-FLAG expression plasmid was generated by inserting cDNA sequences encoding full-length CtIP into the pSP6-FLAG vector (25.Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.-C.W. Hwang L.-Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (626) Google Scholar). CtIP/pSP6-FLAG was then used as template for in vitrosynthesis of radiolabeled CtIP in rabbit reticulocyte lysates (Promega) containing [35S]methionine (ICN). To evaluate the CtIP-specific antibodies, 5-μl aliquots of the programmed lysate were diluted in 500 μl of radioimmune precipitation buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate), incubated with the appropriate antibody reagent for 1 h at 4 °C, and immunoprecipitated as described below. To prepare mammalian cell lysates, HBL-100 and HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum. The cells were lyzed in low salt Nonidet P-40 buffer (10 mm Hepes, pH 7.6, 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA) supplemented with 0.5 mm dithiothreitol, 0.05% SDS, protease inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride) and phosphatase inhibitors (10 mm β-glycerophosphate 5 mm NaF and 0.1 mm vanadate). For immunoprecipitation/co-immunoprecipitation analysis of mammalian cell lysates, the appropriate amount of lysate was co-incubated with the indicated antibodies at 4 °C for 1 h. After adding 50 μl of protein A-Sepharose beads (20% slurry, Amersham Pharmacia Biotech), the mixture was rocked at 4 °C for another 1–3 h. The beads were then washed twice with low salt Nonidet P-40 buffer, twice with high salt Nonidet P-40 buffer (1 m NaCl), and twice again with low salt Nonidet P-40 buffer. Finally, the beads were boiled for 10 min in 30 μl of 2× SDS loading buffer (0.1m Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% β-mercaptoethanol, 0.004% bromphenol blue), and the supernatant was fractionated by electrophoresis. The BR-SZ/pSP6-FLAG expression plasmid, which encodes the C-terminal 336 amino acids of BRCA1 (the "SZ fragment," BRCA1 residues 1528–1863), was generated by inserting BRCA1 cDNA sequences into the pSP6-FLAG vector. BR-SZ/pSP6-FLAG was then used as a template for in vitrosynthesis of the radiolabeled SZ polypeptide in rabbit reticulocyte lysates (Promega) containing [35S]methionine (ICN). Plasmids that encode the various GST-CtIP fusion proteins were generated by inserting appropriate CtIP cDNA sequences into the pGEX-KG expression vector (45.Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). Each GST-CtIP fusion protein was then expressed in E. coli, purified by affinity chromatography on glutathione-agarose beads, and retained as a 50% slurry in buffer C (20 mm Hepes, pH 7.6, 100 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 20% glycerol) containing protease inhibitors. For each GST pull-down assay, a 6-μl aliquot of the radiolabeled SZ fragment was mixed with 60 μl of glutathione-agarose beads (loaded with 20 μg of the appropriate GST-CtIP fusion protein) and 434 μl of radioimmune precipitation buffer. Following a 1-h incubation at room temperature, the beads were washed four times with radioimmune precipitation buffer. The bound SZ polypeptides were eluted by boiling the beads for 10 min in 25 μl of loading buffer (100 mm Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% β-mercaptoethanol, and 0.004% bromphenol blue). The beads were then pelleted by centrifugation, and the supernatant was analyzed by SDS-PAGE. Plasmids encoding the various GAL4p and Vp16 hybrid polypeptides were constructed by inserting cDNA sequences into the pCMV-GAL4 and pVP-FLAG5 expression vectors, respectively (25.Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.-C.W. Hwang L.-Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (626) Google Scholar). Mammalian expression plasmids encoding wild type or mutant (C61G) versions of full-length BRCA1 were generated by inserting the corresponding cDNA sequences into the pCB6 vector. The mammalian two-hybrid assays were conducted in 293 cells as described (46.Tsan J.T. Wang Z. Jin Y. Hwang L.-Y. Bash R.O. Baer R. Bartel P.L. Fields S. The Yeast Two-hybrid System. Oxford University Press, Oxford1997: 217-232Google Scholar). T24 cells were synchronized and analyzed as described (47.Jin Y. Xu X.L. Yang M.-C.W. Wei F. Ayi T.-C. Bowcock A.M. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12075-12080Crossref PubMed Scopus (157) Google Scholar). Western analyses were conducted using 120 μg of cell lysate for BRCA1 immunoblots and 50 μg for CDK2, cyclin A, and CtIP immunoblots. In addition, total RNA was extracted from cells harvested at each time point, and 20-μg aliquots of RNA were evaluated by Northern filter hybridization. Each filter was hybridized in succession with radiolabeled cDNA probes for CtIP, BRCA1, cyclin A, or glyceraldehyde-3-phosphate dehydrogenase using ExpressHyb solution (CLONTECH). The intensity of each autoradiographic signal was then evaluated with Imagequant software (Molecular Dynamics). Whole cell lysates and the membranous, cytoplasmic, and nuclear fractions were prepared from T24 cells as described (47.Jin Y. Xu X.L. Yang M.-C.W. Wei F. Ayi T.-C. Bowcock A.M. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12075-12080Crossref PubMed Scopus (157) Google Scholar). Equivalent volumes of each fraction (corresponding to 30 μg of whole cell lysate) were evaluated by direct immunoblotting with the CtIP-specific monoclonal antibody 14-1 or with monoclonal antibodies that recognize NuMA or α-tubulin (Santa Cruz). For detection of BRCA1, equivalent volumes of each fraction (corresponding to 200 μg of whole cell lysates) were immunoprecipitated with a BRCA1-specific antiserum (25.Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.-C.W. Hwang L.-Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (626) Google Scholar), and the immunoprecipitates were immunoblotted with the BRCA1-specific MS110 monoclonal antibody (Oncogene Research Products) (9.Scully R. Ganesan S. Brown M. Caprio J.A.D. Cannistra S.A. Feunteun J. Schnitt S. Livingston D.M. Science. 1996; 272: 123-125Crossref PubMed Scopus (71) Google Scholar). Approximately 1 × 107 cells were seeded onto each 150-mm culture dish. When the cultures reached 50–70% confluence (usually about 24 h after plating), the cells were subjected to genotoxic stress. For UV irradiation, six dishes of cells were washed with warm phosphate-buffered saline and exposed to 10 J/m2 UV light in a Stratalinker (Stratagene). The irradiated cells were then supplied with fresh tissue culture media and allowed to recover at 37 °C for 1 h before harvest. For adriamycin treatment, four dishes of cells were provided with fresh tissue culture medium containing 0.2 μg/ml adriamycin (Sigma) and incubated at 37 °C for 24 h before harvest. For hydrogen peroxide treatment, six dishes of cells were provided with fresh tissue culture medium containing 10 mmhydrogen peroxide (48.Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (453) Google Scholar). After incubating at 37 °C for 15 min, the cells were washed twice with warm phosphate-buffered saline, supplied with fresh tissue culture medium without hydrogen peroxide, and incubated at 37 °C for an additional 1 h before harvest. To detect CtIP, 50-μg aliquots of the harvested cell lysate were subjected to Western analysis with the CtIP-specific 14-1 monoclonal antibody. To detect p53 and actin, 10-μg aliquots of the same lysates were immunoblotted with the appropriate antibody reagent (Santa Cruz). One-mg aliquots of the lysates were used to detect changes in the mobility of the p220 BRCA1 polypeptide (11.Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). We had previously shown that exogenous CtIP molecules associate in vivo with the endogenous BRCA1 polypeptides of mammalian cells (27.Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). To establish whether endogenous CtIP also interacts with endogenous BRCA1, it was necessary to generate immunological reagents that specifically recognize CtIP. Therefore, three distinct segments of CtIP were expressed in E. coli as fusion proteins with GST (see "Experimental Procedures"). Each fusion protein was purified and used as an immunogen to generate either polyclonal or monoclonal antibodies. The antibody reagents were then tested for immunoprecipitation of full-length CtIP polypeptides synthesized byin vitro translation in the presence of [35S]methionine. Fig. 1shows that radiolabeled CtIP, which migrates with an apparent molecular weight of ∼120 kilodaltons upon SDS-PAGE, was immunoprecipitated with rabbit antisera raised against sequences from either the N-terminal (antiserum 164; CtIP residues 58–369) or C-terminal (antiserum 210; CtIP residues 690–897) halves of CtIP (lanes 2 and7, respectively) but not with the corresponding pre-immune sera (lanes 1 and 6). Radiolabeled CtIP was also immunoprecipitated by a mouse monoclonal antibody raised against CtIP residues 620–897 (antibody 14-1) (lane 12). As expected, immunoprecipitation of 35S-labeled CtIP by antiserum 210 and monoclonal antibody 14-1, both of which recognize C-terminal sequences of CtIP, was blocked by an excess of the GST-CtIP(620–897) fusion protein (lanes 10 and 14) but not GST alone (lanes 8 and 13) or the GST-CtIP(58–369) fusion protein (lane 9). Likewise, immunoprecipitation of radiolabeled CtIP with antiserum 164, which was raised against an N-terminal segment of CtIP, was blocked by an excess of the immunogen, GST-CtIP(58–369) (lane 4) but not by GST alone (lane 3) or GST-CtIP(620–897) (lane 5). These results indicate that the each of the three antibody reagents recognizes CtIP in a highly specific manner. The rabbit antisera (164 and 210) were then used to immunoprecipitate endogenous CtIP from lysates of HBL-100 cells, an immortalized line of normal human mammary epithelial cells (49.Caron de Fromentel C. Nardeux P.C. Soussi T. Lavialle C. Estrade S. Carloni G. Chandrasekaran K. Cassingena R. Exp. Cell Res. 1985; 160: 83-94Crossref PubMed Scopus (95) Google Scholar). The immunoprecipitates were fractionated by SDS-PAGE, and the presence of CtIP in each immunoprecipitate was determined by immunoblotting with the CtIP-specific monoclonal antibody (14-1). As illustrated in Fig.2, a single endogenous CtIP band of ∼120 kilodaltons was detected in an untreated lysate of HBL-100 cells (lane 1). A band with the same electrophoretic mobility was also obtained by immunoprecipitation with CtIP-specific antisera 164 and 210 (lanes 3 and 7, respectively) but not with the corresponding pre-immune sera (lanes 2 and6, respectively). As expected, immunoprecipitation of endogenous CtIP by antiserum 164 was blocked by an excess of the immunogen, GST-CtIP(58–369) (lane 4), but not by GST alone (lane 5). The efficiency of immunoprecipitation with these antisera can be estimated by comparing the intensities of the CtIP bands obtained by immunoblotting the untreated HBL-100 lysate (lane 1) with those obtained by immunoblotting the HBL-100 immunoprecipitates (lanes 3 and 7). As seen in Fig. 2, approximately 3-fold more CtIP is observed in the immunoprecipitates (lanes 3 and 7) than in 0.1 mg of the untreated HBL-100 cell lysate (lane 1). Since the immunoprecipitates were each derived from 0.3 mg of HBL-100 cell lysate, it could be determined that under these conditions, both antisera immunoprecipitate endogenous CtIP with an efficiency that approaches 100%. The CtIP-specific antibodies were then used to test whether endogenous CtIP and BRCA1 polypeptides interact in vivo. Fig. 3 illustrates a co-immunoprecipitation experiment using a lysate of HBL-100 cells. An aliquot of the lysate (2.5 mg of total cellular protein) was immunoprecipitated with CtIP-specific antiserum 210 (lane 3), and smaller aliquots of the same lysate (0.4 mg) were immunoprecipitated with a BRCA1-specific antiserum (lane 5) or a BARD1-specific antiserum (lane 7). The immunoprecipitates were then fractionated by SDS-PAGE, and the presence of BRCA1 in each immunoprecipitate was determined by Western analysis with a BRCA1-specific monoclonal antibody. As expected, BRCA1 was immunoprecipitated with the BRCA1-specific antiserum (lane 5) and co-immunoprecipitated with the BARD1-specific antiserum (lane 7). Significantly, BRCA1 was also co-immunoprecipitated with the CtIP-specific antiserum (lane 3), but not with the corresponding pre-immune serum (lane 2), confirming that the endogenous BRCA1 and CtIP polypeptides are associated in vivo. Given that the CtIP-specific antisera immunoprecipitate CtIP polypeptides from these lysates quantitatively (Fig. 2), it is possible to estimate the fraction of cellular BRCA1 that exists in a CtIP-bound state. As seen in Fig. 3 A, the intensity of the BRCA1 band co-immunoprecipitated with the CtIP-specific antiserum (lane 3) is approximately 1.5-fold higher than that of the BRCA1 band obtained from 0.2 mg of untreated cell lysate (lane 1). Since the co-immunoprecipitated BRCA1 in lane 3 was derived from 2.5 mg of cell lysate, it can be calculated that roughly one-eighth of the cellular BRCA1 pool is bound to CtIP in unsynchronized HBL-100 cells. In contrast, a comparison of the intensities of the BRCA1 bands in lanes 1 and 7suggests that most BRCA1 polypeptides are associated with BARD1 in HBL-100 cells. A reciprocal co-immunoprecipitation experiment is illustrated in Fig.3 B. In this case, HBL-100 lysates (1.2 mg of total cellular protein) were immunoprecipitated with the BRCA1-specific antiserum, and smaller aliquots of the same lysate (0.36 mg) were immunoprecipitated with CtIP-specific antiserum 210. As shown, end
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