Pag, a Putative Tumor Suppressor, Interacts with the Myc Box II Domain of c-Myc and Selectively Alters Its Biological Function and Target Gene Expression
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m206066200
ISSN1083-351X
AutoresZ M Mu, Xiao Ying Yin, Edward V. Prochownik,
Tópico(s)Melanoma and MAPK Pathways
ResumoThe highly conserved Myc Box II (MBII) domain of c-Myc is critically important for transformation and transcriptional regulation. A yeast two-hybrid screen identified Pag as a MBII-interacting protein. Pag, a member of the peroxiredoxin family, has been reported previously to bind to and inhibit the cytostatic properties of the c-Abl oncoprotein. We now show that Pag promotes increased cell size and confers a proapoptotic phenotype, two hallmark features of ectopic c-Myc overexpression. Pag and c-Myc also confer resistance to oxidative stress, a previously unrecognized property of the latter protein. In contrast, Pag inhibits tumorigenesis by c-Myc-overexpressing fibroblasts and causes a broad but selective loss of c-Myc target gene regulation. Pag is therefore an MBII-interacting protein that can either mimic or enhance some of the c-Myc properties while at the same inhibiting others. These features, along with the previously identified interaction with c-Abl, provide support for the idea that Pag functions as a tumor suppressor. The highly conserved Myc Box II (MBII) domain of c-Myc is critically important for transformation and transcriptional regulation. A yeast two-hybrid screen identified Pag as a MBII-interacting protein. Pag, a member of the peroxiredoxin family, has been reported previously to bind to and inhibit the cytostatic properties of the c-Abl oncoprotein. We now show that Pag promotes increased cell size and confers a proapoptotic phenotype, two hallmark features of ectopic c-Myc overexpression. Pag and c-Myc also confer resistance to oxidative stress, a previously unrecognized property of the latter protein. In contrast, Pag inhibits tumorigenesis by c-Myc-overexpressing fibroblasts and causes a broad but selective loss of c-Myc target gene regulation. Pag is therefore an MBII-interacting protein that can either mimic or enhance some of the c-Myc properties while at the same inhibiting others. These features, along with the previously identified interaction with c-Abl, provide support for the idea that Pag functions as a tumor suppressor. basic helix-loop-helix leucine zipper transactivation domain Myc Box I/II glutathione S-transferase Dulbecco's modified minimal essential medium fetal bovine serum chloramphenicol phosphate-buffered saline polyvinylidene difluoride terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling expressed sequence tag The c-MYC oncogene product influences many cellular processes, including growth, cell cycle progression, apoptosis, and differentiation (1Henriksson M. Lüscher B. Adv. Cancer Res. 1996; 68: 109-181Crossref PubMed Google Scholar, 2Obaya A. Mateyak M.K. Sedivy J.M. Oncogene. 1999; 18: 2934-2941Crossref PubMed Scopus (201) Google Scholar, 3Prendergast G.C. 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Oncogene. 1999; 18: 2942-2954Crossref PubMed Scopus (153) Google Scholar). Two proteins, Bin-1 and TRRAP, have been the most intensely studied in this regard (27Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (312) Google Scholar, 28McMahon S.B. Van Buskirk H.A. Dugan K.A. Copeland T.D. Cole M.D. Cell. 1998; 94: 363-374Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). Both interact with a subregion of the TAD, termed "Myc Box II" (MBII) that is highly conserved among all Myc oncoprotein members. MBII is required for c-Myc to properly regulate many of its target genes and to transform fibroblasts in vitro in association with activated ras oncogenes (29Stone J. DeLange T. Ramsay G. Jakobovits E. Bishop J.M. Varmus H. Lee W. Mol. Cell. Biol. 1987; 7: 1697-1709Crossref PubMed Scopus (331) Google Scholar,30Nesbit C.E. Tersak J.M. Grove L.E. Drzal A. Choi H. Prochownik E.V. Oncogene. 2000; 19: 3200-3212Crossref PubMed Scopus (55) Google Scholar). Bin-1 appears to be a negative regulator of c-Myc transformation, whereas TRRAP appears to be a positive regulator (27Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (312) Google Scholar, 28McMahon S.B. Van Buskirk H.A. Dugan K.A. Copeland T.D. Cole M.D. Cell. 1998; 94: 363-374Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). The mechanism by which Bin-1 represses transformation appears complex, but includes effects on the cell cycle and the promotion of apoptosis in response to c-Myc over-expression (26Sakamuro D. Prendergast G.C. Oncogene. 1999; 18: 2942-2954Crossref PubMed Scopus (153) Google Scholar). An important aspect of TRRAP1 function may involve its role as a docking site for a complex of proteins that includes one or more of the histone acetylases discussed above (11McMahon S.B. Wood M.A. Cole M.D. Mol. Cell. Biol. 2000; 20: 556-562Crossref PubMed Scopus (374) Google Scholar). We utilized a yeast two-hybrid approach to screen for proteins that interact with the c-Myc TAD. We describe one such protein, Pag, also known as MSP23 and peroxiredoxin-1. Pag is a 23-kDa protein whose cDNA was originally cloned from ras oncogene-transformed mammary epithelial cells (31Prosperi M.-T. Ferbus D. Karczinski I. Goubin G. J. Biol. Chem. 1993; 268: 11050-11056Abstract Full Text PDF PubMed Google Scholar). It is up-regulated by serum and by compounds that induce oxidative stress (32Prosperi M.-T. Ferbus D. Rouillard D. Goubin G. FEBS Lett. 1998; 423: 39-44Crossref PubMed Scopus (42) Google Scholar). Pag has also been shown to interact with the Src homology-3 and kinase domains of c-Abl and to reverse the cytostatic effects of c-Abl (33Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar). In the current work, we show that Pag also interacts specifically with the MBII region of the c-Myc TAD both in vitro and in vivo. Pag overexpression promotes a marked increase in cell size and enhances apoptosis, thus mimicking two previously described c-Myc phenotypes. We also show that Pag and c-Myc are each able to protect cells from apoptosis induced by oxidative damage. In contrast, Pag inhibits anchorage-independent growth of c-Myc overexpressing fibroblasts and down-regulates some, but not all, c-Myc target genes. Our results identify Pag as an MBII-binding protein that can differentially regulate certain c-Myc-dependent functions. The ability of Pag to interact with and modulate the activities of both c-Myc and c-Abl strongly supports its candidacy as a tumor suppressor. Standard polymerase chain reactions (PCR) were used to amplify the indicated murine c-Myc sequences from previously described c-Myc expression vectors (30Nesbit C.E. Tersak J.M. Grove L.E. Drzal A. Choi H. Prochownik E.V. Oncogene. 2000; 19: 3200-3212Crossref PubMed Scopus (55) Google Scholar). All PCR primers contained BamHI restriction sites in the 5′ (forward)-primer and BglII sites in the 3′ (reverse)-primer. The initial "bait" plasmid consisted of an ADH1 promoter-driven fusion between codons 1 and 1067 of the human Sos guanyl nucleotide exchange factor and murine c-myc codons 2–147, which encode the TAD. Additional baits consisted of TAD mutants bearing deletions of MBI (codons 43–69), codons 70–130, and MBII (codons 133–147), as well as a full-length wild-type c-myc (codons 2–439). The orientation, sequence, and reading frame of each clone were confirmed by DNA sequencing using a common pSos sequencing primer. pSos plasmids were introduced into the cdc25H yeast strain by the polyethylene glycol/lithium acetate method, and transformants were selected on SD/Leu− + glucose plates at 25 °C. Successful transformation was confirmed by recovery of each input plasmid in theEscherichia coli XL-1 Blue strain. For cDNA library screening, the strain containing pSos-c-Myc (2–147) was expanded in liquid culture and transformed with a human spleen cDNA library unidirectionally cloned into the pMyr vector (Stratagene, La Jolla, CA). Approximately 2 × 106 transformants were initially plated onto SD/Ura−leu− glucose plates and incubated for 2 to 3 days at 25 °C. The barely visible colonies were then replica plates onto SD/Ura−leu− galactose plates and incubated for an addition 5 to 7 days. Approximately 300 colonies >1 mm in size grew under these conditions. For DNA sequencing, 30 pMyr plasmids were recovered in XL-1 Blue cells (Stratagene) by selecting for chloramphenicol resistance. Immunoblotting of yeast lysates was performed as described previously (34Langlands K. Prochownik E.V. Anal. Biochem. 1997; 249: 250-252Crossref PubMed Scopus (15) Google Scholar) using a monoclonal antibody against the Sos protein (Transduction Laboratories, Lexington, KY). All pSos and pMyr plasmids were subjected to DNA sequencing on an ABI373 automated sequencer (Applied Biosystems, Foster City, CA). Sequencing primers were synthesized to regions ∼150 bp upstream of the polylinker region of each plasmid. All pMyr clones derived from the spleen cDNA library were subjected to GenBankTM BLAST searches, which were used to establish the identity and reading frame of each insert sequence. PCR was used to amplify the region between codons 2 and 219 of either wild-type c-Myc or a deletion MBII mutant. The common 5′ PCR primer contained an engineered EcoRI site, and the 3′ PCR primer contained aBamHI site. PCR products were digested withBamHI, blunt-ended with the Klenow fragment of DNA polymerase I, digested with EcoRI, and unidirectionally cloned into the EcoRI + XhoI (blunt-ended) digested pGEX-4T vector (Amersham Biosciences). The orientation and reading frame of each insert was confirmed by DNA sequencing. In all cases, GST fusion proteins of the predicted sizes were induced and purified by glutathione-agarose affinity chromatography as described previously (35Gupta K. Anand G. Yin X.Y. Prochownik E.V. Oncogene. 1998; 16: 1149-1159Crossref PubMed Scopus (22) Google Scholar). A Pag cDNA encoding the full-length protein was excised from the original pMyr yeast vector, recloned in pBluescript-SK (Stratagene), and used in a coupled T3 RNA polymerase in vitro transcription/translation reaction in a rabbit reticulocyte lysate system (Promega, Madison, WI). One-tenth of the reaction (5 μl) was mixed with 1 μg of each of the above GST proteins in 0.5 ml of NETN buffer (100 mm NaCl, 20 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 0.5% Nonidet P-40 detergent (Sigma)). After 2 h of incubation at 4 °C with continuous agitation, 20 μl of NETN-washed glutathione-coupled agarose (Bio-Rad) was added, and mixing was continued for an additional 2 h at 4 °C. Precipitates were collected by low speed centrifugation and washed five times in NETN buffer before being resuspended and boiled in SDS-PAGE buffer. Electrophoresis was performed on standard SDS-12% polyacrylamide gels followed by autoradiography of the dried gel (XAR film, Kodak, Rochester, NY). NIH3T3 and COS-7 cells were routinely cultured in Dulbecco's modified minimal essential medium (D-MEM, Invitrogen) supplemented with 2 mm glutamine, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 10% supplemented calf serum (all from Invitrogen). Rat1a fibroblasts were cultured identically except that 10% fetal bovine serum (FBS, Invitrogen) was used. 32D myeloid cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS and 10% conditioned medium from the interleukin-3-producing WEHI-3B cell line. ST486 Burkitt's lymphoma cells were cultured in RPMI plus 10% FBS. All cultures were split at least twice weekly to maintain continuous logarithmic growth. Soft agar colony assays for Rat1a transfectants were performed in 60-mm tissue culture plates by mixing 4 × 103 cells in 2 ml of 0.35% molten agarose (Invitrogen) in D-MEM, 10% FBS. The mixture was plated on a 4-ml solidified layer of 0.7% agarose prepared in the same medium. The number and sizes of the resulting colonies were determined by microscopic visualization 12–14 days later. For tumorigenesis assays, 3–4-week-old nude mice were obtained from Charles River Laboratories (Wilmington, MA). Five animals were used in each group. Each animal was inoculated subcutaneously with 107 Rat1a fibroblasts resuspended in 0.1 ml of D-MEM without serum. Tumor growth was monitored twice weekly. The mammalian expression vectors pSVL-neo, pSVL-puro, pSVL-neo-c-Myc, pSVL-neo-c-Myc(delMBI), and pSVL-neo-c-Myc(delMBII) have been described previously (30Nesbit C.E. Tersak J.M. Grove L.E. Drzal A. Choi H. Prochownik E.V. Oncogene. 2000; 19: 3200-3212Crossref PubMed Scopus (55) Google Scholar). Pag was expressed as a C-terminal Myc epitope-tagged (MT) protein either in the pSVL-puro vector or in the pCBF vector under the control of a cytomegalovirus early promoter (gift from M. Cole, Princeton University). Plasmid DNAs were purified on Qiagen columns (Qiagen, Chatsworth, CA) according to the directions of the supplier. Transient transfections using supercoiled DNAs were performed with LipofectAMINE reagent (Invitrogen). The indicated cell lines were plated at 8 × 105 cells/dish in 100-mm tissue culture plates 1 day prior to transfection and were harvested 2 days after transfection. In addition to the indicated plasmids, each plate also received 1 μg of pCMVβgal (Clontech, La Jolla, CA). Lysates were assayed for β-galactosidase, luciferase, and CAT activities as previously described (36Zhang H. Fan S. Prochownik E.V. J. Biol. Chem. 1997; 272: 17416-17424Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Stable transfections in NIH3T3 and Rat1a fibroblasts were performed using LipofectAMINE essentially as described above except that 2 days after the addition of DNA, cultures were split 1:5 and selected in either puromycin (1 μg/ml, Sigma) or G-418 (750 μg/ml absolute concentration, Invitrogen). After 10–14 days, well isolated colonies were picked and expanded for further characterization. Stable transfections in 32D cells were performed by electroporation with linearized plasmid DNAs, selected 2 days later in the appropriate drug, and either pooled or cloned by limiting dilution in 96-well plates. For co-immunoprecipitation from transiently transfected cells, 50% confluent 100-mm duplicate plates of COS-7 cells were each transfected with 5 μg of the pSVLneo-c-Myc plasmid, the pSVLneo-c-Myc(delMBI) plasmid, or the pSVL-neo c-Myc(delMBII) plasmid and 3 μg of pSVLpuro-MT-Pag plasmid using LipofectAMINE. Control transfections consisted of the same amounts of each of the individual plasmids described above plus pSVLneo or pSVLpuro parental plasmids. 48 h later, cells were collected by scraping, washed three times in PBS, and resuspended in 0.32 m sucrose, 10 mm Tris-HCl, pH 7.5, 5 mm MgCl2, and 1% Triton-X100 (Sigma) containing protease inhibitor mixture (Roche Molecular Biochemicals). After 10 min on ice, nuclei were concentrated by low speed centrifugation, washed twice, and resuspended in 1 ml of the same buffer. They were then disrupted on ice by a 30-s pulse with the microtip of a Branson sonifier at a setting of 7. The lysate was cleared by centrifugation at 10,000 × g for 5 min followed by an additional round of clearing for 2 h at 4 °C with 10 μl of control IgG followed by 50 μl of protein A-agarose (BioRad). After centrifugation, 10 μl of rabbit anti-c-Myc antibody prepared against amino acids 1–262 of the human protein (sc-764, Santa Cruz Biotechnology) was added and incubated at room temperature for 2 h with constant agitation. Immune complexes were precipitated by the addition of 50 μl of protein A-agarose for 2 h. After extensive washing, the complexes were resuspended in 50 μl of SDS-PAGE lysing buffer, boiled, and resolved by SDS-12% PAGE. After semidry transfer to a PVDF membrane (Millipore, Bedford, NY), the membrane was blocked for 2 h with PBS, 0.1% Tween 20 (PBS-T) containing 5% dry milk. The membrane was then divided in two. The upper half was incubated with a 1:500 dilution of the above mentioned polyclonal anti-c-Myc antibody, and the lower portion was probed with a 1:500 dilution of the 9E10 anti-c-Myc epitope-tagged monoclonal antibody (sc-40, Santa Cruz). Both blots were incubated overnight at 4 °C. After extensive washing in PBS-T, the first blot was incubated for 2 h at room temperature with a 1:500 dilution of horseradish peroxidase-conjugated goat-anti rabbit IgG (Santa Cruz), and the second blot was incubated identically with a 1:500 dilution of horseradish peroxidase-conjugated rabbit-anti mouse IgG. After washing in PBS-T, both blots were developed using an enhanced chemiluminescence kit (Renaissance Kit, PerkinElmer Life Sciences) according to the supplier's directions. For co-immunoprecipitation of endogenous c-Myc and Pag proteins, 2 × 107 cells from the ST486 Burkitt's lymphoma cell line were lysed and sonicated as described above. The supernatant was then cleared by the addition a 1:200 dilution of control goat serum for 2 h at room temperature followed by 50 μl of staphylococcus A-agarose for an additional 2 h. The supernatant was then incubated with a 1:200 dilution of either control goat IgG or a goat anti-Pag IgG (sc-7380, Santa Cruz) for 2 h at room temperature followed by the addition of 50 μl of protein-A agarose. The precipitates were washed three times with 1 ml of lysis buffer, divided into two equal fractions, boiled in running buffer, and resolved by 10% SDS-PAGE. After Western transfer, one of the duplicate PVDF membranes was probed with a 1:500 dilution of the 9E10 anti-c-Myc monoclonal antibody and the second membrane was probed with a control antibody. Both blots were then developed by chemiluminescence. 3T3-neo or 3T3-Pag cell lines were grown in 100-mm plates to ∼80% confluency. To determine the total protein content, triplicate cultures were harvested individually by scraping, and the total number of cells was determined by manual counting using a hemacytometer. Equivalent numbers of cells were then pelleted by low speed centrifugation, lysed, and the protein content determined using BCA reagent (Pierce). The rate of protein synthesis was determined by washing an equivalent set of triplicate plates three times in PBS followed by the addition of 3 ml of cysteine + methionine-free D-MEM (ICN Biomedicals, Costa Mesa, CA). After a 30-min incubation at 37 °C, 35S-labeled cysteine + methionine (Easy-tag protein labeling mix; specific activity, 1175 Ci/mmol, PerkinElmer Life Sciences) was added to a final concentration of 200 μCi/ml for 1 h. The cells were then harvested by scraping, washed three times in PBS, and lysed for 30 min in 1 ml of 10 mm Tris, pH 8.0, 1% SDS, plus protease inhibitor mixture. 10 μl of each lysate + 20 μg of carrier bovine serum albumin was precipitated by the addition of 1 ml of ice-cold 10% trichloroacetic acid. The precipitates were collected on 25-mm GF/A glass fiber papers (Whatman), washed exhaustively with cold 10% trichloroacetic acid, dried, and subjected to scintillation counting. Flow cytometric analysis of propidium iodide-stained nuclei was performed on a BD Pharmingen FACSTAR fluorescence-activated cell sorter (8Yin X.Y. Grove L. Datta N. Long M.W. Prochownik E.V. Oncogene. 1999; 18: 1177-1184Crossref PubMed Scopus (124) Google Scholar). 2 × 104 cells were analyzed for each assay. Quantitation was performed using single histogram statistics. To identify proteins that interact with the c-Myc TAD, we conducted a yeast two-hybrid screen using the TAD (amino acids 2–147) as the bait. Because this region is highly self-transactivating in conventional two-hybrid assays, 2Z.-M. Mu and E. V. Prochownik, unpublished observation. we utilized the CytoTrapTM system (Stratagene) in which interaction between bait and target proteins reconstitutes the Ras signaling pathway in Saccharomyces cerevisiae. A yeast strain expressing the c-Myc TAD in the pSos vector was transformed with a pMyr human spleen cDNA expression library, and 2 × 106 colonies were screened for growth at 37 °C. 30 pMyr plasmids were recovered for DNA sequencing, which showed that six encoded in-frame fusions with Pag. Of these, three initiated at different positions within the 5′-untranslated region or within five codons of the Pag translational start site. Five other pMyr isolates contained in-frame fusions with a cDNA encoding the previously described c-Myc-interacting protein, MM-1 (37Mori K. Maeda Y. Kitaura H. Taira T. Iguchi-Ariga T. Ariga H. J. Biol. Chem. 1998; 273: 29794-29800Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). We initially verified the specificity of the observed interaction between c-Myc and Pag in yeast. One of the full-length pMyr-Pag plasmids was co-expressed with the original pSos-c-Myc (2–147) TAD vector, pSos encoding a fusion with full-length c-Myc (pSos-c-Myc-(2–439)), or a series of pSos-c-Myc fusions containing deletions within the c-Myc TAD (30Nesbit C.E. Tersak J.M. Grove L.E. Drzal A. Choi H. Prochownik E.V. Oncogene. 2000; 19: 3200-3212Crossref PubMed Scopus (55) Google Scholar). These deletions included Myc Box I (MBI, codons 43–69), codons 70–130, and MBII (codons 133–147). Two additional yeast strains served as independent positive and negative controls for growth at 37 °C. The first contained pSos and pMyr plasmids, each of which expressed the full-length MafB protein. The second contained a pSos-collagenase IV fusion protein and a pMyr-lamin C fusion protein. After selection of Leu + Ura prototrophs at 25 °C, several colonies of each strain were replated and grown at 37 °C. As seen in Fig. 1, yeast containing either the original pSos-c-Myc (2–147) bait or the full-length c-Myc protein readily grew at 37 °C in the presence of co-expressed Pag (lines 5 and 6), thus confirming the original finding as well as indicating that interaction between c-Myc and Pag occurred with the full-length forms of both proteins. Deletions of either MBI or of the region between MBI and MBII (amino acids 70–130) still permitted strong interaction with Pag (lines 7 and8). However, deletion of MBII abolished the interaction (line 9). As expected, growth at 37 °C was seen with yeast harboring the MafB fusion proteins (line 2), whereas no growth was seen in yeast expressing only a single fusion protein (lines 3 and 4) or the pSos-collagenase and pMyr-lamin C combination (line 1). Finally, growth at 37 °C was seen only when the yeast were propagated on galactose-containing plates, thus verifying that both fusion proteins need to be expressed. To confirm the validity of the observed interactions, several of the above yeast strains were propagated in liquid culture and examined by immunoblotting (34Langlands K. Prochownik E.V. Anal. Biochem. 1997; 249: 250-252Crossref PubMed Scopus (15) Google Scholar) using a monoclonal antibody directed against Sos. As seen in Fig. 1 C, each of the yeast strains expressed a pSos fusion protein of the predicted size. These results indicated that the failure of Pag to interact with the MBII-deleted form of c-Myc was not because of lack of expression of the latter protein. To verify independently the interaction between Pag and c-Myc, we expressed Pag as a 35S-labeled in vitro translation product and performed pull-down experiments with affinity-purified GST fusion proteins containing the first 219 amino acids of c-Myc (GST-c-Myc-(2–219)), the same region containing a MBII deletion (GST-c-Myc-(2–219)delMBII) or GST alone. As seen in Fig.2, a significant portion of the labeled Pag protein interacted with the first fusion protein but with neither of the latter two. These results confirm those of the yeast two-hybrid experiments described above indicating that an intact c-Myc MBII is necessary for the interaction with Pag in vitro. Preliminary Northern and RNA "dot blot" experiments, as well as Western blots, indicated that Pag is ubiquitously expressed in mammalian cells and tissues, with the highest levels occurring in liver and kidney (not shown). To demonstrate the c-Myc-Pag interaction in mammalian cells, we first co-expressed Pag as a c-Myc-epitope-tagged fusion protein in COS-7 cells along with either full-length, wild-type c-Myc or MBI or MBII c-Myc deletions (30Nesbit C.E. Tersak J.M. Grove L.E. Drzal A. Choi H. Prochownik E.V. Oncogene. 2000; 19: 3200-3212Crossref PubMed Scopus (55) Google Scholar). c-Myc proteins were immunoprecipitated from lysates using an antibody directed against the N-terminal half of the protein. Western blots were then probed with the same antibody to confirm expression of the c-Myc proteins, or with the 9E10 monoclonal antibody directed against the Pag c-Myc epitope tag, which is derived from the extreme C terminus of c-Myc. As seen in Fig.3 A, Pag was detected when it was co-expressed with either wild-type (WT) c-Myc or with c-MycdelMBI. In the absence of co-expressed c-Myc or in the presence of c-Myc delMBII, no Pag protein was co-immunoprecipitated. In other experiments, we asked whether endogenous c-Myc and Pag could be co-immunoprecipitated. Preliminary studies determined that the ST486 Burkitt's lymphoma cell line expressed high levels of both proteins (not shown). Therefore, a lysate from this cell line was incubated with an anti-pag antibody or with an equivalent amount of a control antibody. Following the addition of protein A-agarose, the precipitates were divided in two, and each was resolved by SDS-PAGE and blotted to a PVDF membrane. One of the blots was then incubated with the
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