Monoclonal Antibodies - Molecular and Cellular Biology - American ...

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Nov 20, 1987 - We have generated monoclonal antibodies by using a synthetic peptide corresponding ... The monoclonal antibodies not only identified the fos.
MOLECULAR AND CELLULAR BIOLOGY. May 1988, p. 2251-2256 0270-7306/88/052251-06$02.00/0 Copyright t 1988, American Society for Microbiology

Vol. 8. No. 5

Detection of fos Protein during Osteogenesis by Monoclonal Antibodies PIETRO DE TOGN1,1 HENRY NIMAN, VINCENT RAYMOND.1 PAUL SAWCHENKO.1 AND INDER M. VERMA1* The Salk Instititte, P. 0. Box 85800, Sanz Diego, California 92138,1 (1itd S(cripps Clinic anid Resear chl Foild(ationl, La Jolla, California 920372 Received 20 November 1987/Accepted 9 February 1988

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

described elsewhere (21, 22), except that SP2/0 myeloma cells were used. Briefly, synthetic peptides were coupled to keyhole limpet hemocyanin by using in-maleimido-benzoylN-hydrosuccinimide as the coupling agent (16). The conjugate was diluted to 1 mg/ml with phosphate-buffered saline (PBS) at pH 7.4, and it was mixed with an equal volume of complete Freund adjuvant. Each mouse (strain 129G1X+) was immunized intraperitoneally with 50 p.g of peptide conjugate on day 0. On day 144, an equal amount of peptide mixed with an equal volume of alum (10 mg/ml) was injected intraperitoneally. Peptide in PBS was administered intravenously on day 186. The spleen was removed on day 189. Hybridomas were formed by fusing the spleen cells with SP2/0 nonsecreting myeloma cells by using polyethylene glycol (20). The hybridomas were screened by enzymelinked immunosorbent assay (21). After at least two subclonings by limiting dilutions, the cells were expanded, and approximately 107 cells were injected into the peritoneal cavity of pristane-primed 129G1X+ x BALB/c mice. Only monoclonal antibodies found positive to peptide by enzymelinked immunosorbent assay were further tested. Monoclonal antibodies raised against the amino-terminal synthetic peptide immunoprecipitated 35S-labeled c-fos protein from HeLa cells induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) (Fig. 1). Briefly, the nuclear extracts used in these experiments were prepared as follows. HeLa cells growing in suspension were harvested by centrifugation and suspended at a concentration of 2 x 106 cells per ml in methionine-free Dulbecco modified Eagle medium with 10% dialyzed fetal calf serum. [35S]methionine and 50 ng of TPA per ml were added, and the incubation was continued for another 45 min. The cells were then harvested and suspended in hypotonic buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 1.5 mM MgCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin; pH 7.5) and kept at 4°C for 10 min. The cells were lysed by the addition of 0.2% Nonidet P-40, and nuclei were suspended in 2 ml of lysis buffer (50 mM Tris, 0.5% sodium dodecyl sulfate, 70 mM 3-mercaptoethanol; pH 8.0). The extracts were diluted in RIPA buffer without sodium dodecyl sulfate (10 mM Tris hydrochloride, 150 mM NaCl, 1% deoxycholate, 1% Nonidet P-40, 1% aprotinin, 1 mM phen-

Proto-oncogene fos has been implicated in cell growth, differentiation, and development (18, 27, 28). Extensive analysis of its expression suggests both stage and tissue specificities of the gene, but its direct role in cellular metabolism remains elusive. Recently, however, three different lines of experimental observations offer clues to the function of the fos protein: (i) the retardation of cell growth and cell proliferation by the use of antisense fos recombinant constructs (12, 23), (ii) the presence of fos protein in transcriptional complexes regulating adipocyte differentiation (6), and (iii) the effect on bone development of overexpression of fos in transgenic mice (24). A role for the fos gene in bone formation is further substantiated by the identification of c-fos transcripts in the growth regions of fetal bone and mesodermal web tissue (7). The expression of the c-fos gene in the bone tissue is particularly intriguing because FBJmurine osteosarcoma virus containing the v-fos gene induces bone tumors (8). Proto-oncogenefos is induced by a large number of stimuli ranging from mitogens, differentiation-specific agents, pharmacological agents, stress, etc. (11, 14, 28a). Induction is almost invariably very rapid but transient, and at maximal induction, the c-fos protein represents about 0.01% (about 3,000 to 5,000 molecules per cell) of the total cellular protein. The low abundance of the c-fos protein, combined with its short half-life and extensive posttranslational modification, has often made its identification a tedious task (1). We report the characterization of monoclonal antibodies raised against an amino-terminal synthetic fos peptide which could detect both the v- and c-fos proteins. Characterization of monoclonal antibodies. We synthesized synthetic peptides corresponding to the predicted humanfos protein amino-terminal (amino acids 4 to 17, with one serine deletion at position 15 [26]), carboxy-terminal (amino acids 359 to 378), and M-peptide (amino acids 132 to 153) regions by using modifications (13) of the solid-phase synthesis described earlier (17; Fig. 1A). The peptide sequences were confirmed by high-pressure liquid chromatography and amino acid analysis. Monoclonal antibodies were isolated as *

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FIG. 1. Immunoprecipitation of p55C5"' with various monoclonal antibodies. (A) Locations and primary sequences of thefos peptides used to raise the antibodies. The numbers indicate amino acid (aa) residues in the protein. The arrow indicates the serine residue which was deleted in the synthesis of the N peptide. The sequence of the M2 peptide (M-Peptide; amino acids 127 to 152) used for the preparation of the rabbit fos antiserum (aM2) is also shown. (B) Immunoprecipitation of p55C`-J from HeLa cell nuclear extracts. The monoclonal antibodies were grouped on the basis of their specificity for the N peptide (anti-N; lanes 3 to 13), the M peptide (anti-M; lanes 14 to 19), and the C peptide (anti-c; lanes 20 to 25). The control with normal serum (N) is shown in lane 1. The products were analyzed by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel. The numbers on the left indicate the molecular weights (103) of marker proteins. (C) Immunoprecipitation of p55C`t65 from radiolabeled HeLa cells. Nuclei from [35]methionine-labeled HeLa cells were extracted in sodium dodecyl sulfate lysis buffer (SDS; lanes 1 to 3) or in RIPA buffer (RiPA; lanes 4 to 6). Aliquots from each nuclear extract were immunoprecipitated with affinity-purified aM2fos antiserum or with monoclonal antibody 14C1. The syrnbols + and - indicate whether 14C1 was preabsorbed (+) or not (-) with 100 ,ug of N peptide per ml. The relative positions of the p39 and fos proteins are indicated.

ylmethylsulfonyl fluoride) before centrifugation for 60 min at 100,000 x g. Equivalent amounts of nuclear extracts were incubated with affinity-purified aLM2 fos antiserum and ascites fluid from the different hybridomas for 60 min at 4°C. Immunocomplexes were precipitated by adding Formalinfixed Staphylococcus aureus (Pansorbin; Calbiochem), and after 60 min of incubation, the suspension was centrifuged. The pellet in sample buffer was boiled for 3 min, and the superhatant was analyzed on a 10% polyacrylamide gel (15). The gel was dried and processed by fluorography (2). Of the 11 monoclonal antibodies directed against the N-terminal peptide, 10 immunoprecipitated a 55-kilodalton (kDa) protein analogous in size to that observed with M2 antisera (Fig. 1B). We wanted to further characterize some of the monoclofial antibodies and chose 14C1 because it gave the strongest signal (Fig. 1B, lane 3). 14C1 antibodies were also capable of recognizing the p55-p39 cellular protein complex similar to that observed with M2 antisera (5) (Fig. 1C, lane

5). p39 is a cellular protein which is noncovalently bound to p55 and is not observed when the samples are treated with sodium dodecyl sulfate prior to immunoprecipitation (Fig. IC). No 55-kDa protein could be immunoprecipitated when the samples were preincubated with N peptide, indicating that the 14C1 monoclonal antibody is specific forfos protein and is directed against the amino-terminal region of the protein. c-fos protein, as well as v-fos protein, which has a different C terminus (25), was efficiently immunoprecipitated by monoclonal antibody 14C1 (Fig. 2A, lanes 5 and 7). Furthermore, the monoclonal antibodies recognized mouse (lane 3) and rat (lane 9) fos proieins. The c-fos protein appeared to be more modified than the v-fos protein (compare lanes 3 and 5), suggesting that the -14C1 monoclonal antibody can recognize modified forms of thefos protein. No fos protein was detected with 14C1 in TPA-induced chicken embryo fibroblasts (lane 11). However, atM2 antisera

VOL. 8, 1988

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