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DETECTION

OF NGF

RECEPTORS

AFTER GENE TRANSFER

61

Acknowledgments I thank Michael S. Cole, Jeffrey K. Glenn, and Lori B. Taylor for technical contributions, Dennis O. Clegg for NGF/M13 DNA and probe labeling protocols, Paul D. Coleman for tissue from the Rochester Alzheimer's Disease Program, and Paul H. Johnson for critical reading of the manuscript. This work was supported by National Institutes of Health Grant AGO3644 and by SRI International Research and Development funds.

[6] D e t e c t i o n o f N e r v e G r o w t h F a c t o r R e c e p t o r s a f t e r Gene Transfer B y MOSES V . CHAO

Introduction The nerve growth factor (NGF) receptor is a integral membrane glycoprotein, with a single membrane-spanning domain, a negatively charged extracellular domain rich in cysteine residues, and a cytoplasmic domain of 155 amino acids. 1.2 The receptor is encoded by a single-copy gene that gives rise to a single 3.8-kilobase (kb) mRNA. 3 Although the mode of signal transduction for NGF has not been fully clarified, it is probable that an auxiliary protein(s) closely associated with the receptor is necessary to mediate the effects of NGF in responsive c e l l s . 4'5 These effects ultimately result in increases in gene expression, activation of neurotransmitter enzymes, and neurite outgrowth. Further molecular and mechanistic studies of NGF action will depend on rapid and reliable methods of detecting the receptor in order to assess its role in signal transduction. This chapter reviews accessible ways in which cell surface expression of the NGF receptor is determined after gene transfer of the cloned gene. Many of the techniques cited below are applicable to a wide number of receptor systems provided that the ligand and/or monoclonal antibodies are readily available.

1 D. Johnson, T. Lanahan, C. R. Buck, A. Sehgal, C. Morgan, E. Mercer, M. Bothwell, and M. Chao, Cell (Cambridge, Mass.) 47, 445 (1986). 2 M. J. Radeke, T. P. Misko, C. Hsu, L. Herzenberg, and E. M. Shooter, Nature (London) 325, 593 (1987). 3 M. V. Bothwell, A. H. Ross, H. Koprowski, A. Lanahan, C. R. Buck, and A. Sehgal, Science 232, 418 (1986). 4 M. Hosang and E. M. Shooter, J. Biol. Chem. 260, 655 (1985). 5 B. L. Hempstead, L. S. Schleifer, and M. V. Chao, Science 243, 373 (1989).

METHODS IN ENZYMOLOGY, VOL. 198

Copyright © 199l by Academic Press, Inc. All rights of reproduction in any form reserved.

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Gene Transfer DNA-mediated gene transfer of high molecular weight DNA, followed by rescue and cloning of transformed sequences, led to the isolation of the NGF receptor gene.2'3 Monoclonal antibodies directed against the receptor were extremely instrumental in the detection and purification of transfected cell lines. The two most commonly used are the IgG-192 antibody, which recognizes the rat receptor, 6 and the ME20.4 antibody, specific for the human receptor. 7 Neither antibody displays any cross-reactivity, but each can be used selectively to immunoprecipitate labeled receptor, z'3'5 For initial analysis of NGF receptors in cultured cells, in situ rosetting and indirect immunofluorescence provide straightforward and accurate methods. These techniques can be applied to cultured cells or to transfection experiments with the NGF receptor gene. Figure 1 shows the general scheme for detecting stable expression after transfection. A typical eukaryotic expression vector, pMV7, containing murine Moloney sarcoma viral sequences 8 has been employed, owing to its versatility as an double gene vector. An internal promoter from the herpesvirus thymidine kinase gene is used to express the selectable marker, aminoglycoside phosphotransferase, which detoxifies the antibiotic neomycin (G418). The viral 5' long terminal repeat (LTR) is used to transcribe a full-length human receptor cDNA sequence. For a variety of cell types, the calcium phosphate precipitation procedure is widely used, as described in detail by Wigler et al. 9 For some cells such as the rat pheochromocytoma PC12 cell line, stable transformants are difficult to obtain, partly because of the slower rate of cell division and the nonadherent properties of the cells. However, treatment of PC 12 cells with 20% (v/v) glycerol after exposure to transforming DNA 1° enhances the efficiency of transfection. Other methods such as electroporation, lipofectin, and DEAE-dextran can also improve the efficiency of transfection. The availability of stable transformants permits a more detailed analysis of the biochemical and kinetic properties of the ligand and transfected

6 C. E. Chandler, L. M. Parsons, M. Hosang, and E. M. Shooter, J. Biol. Chem. 259, 6882 (1984). 7 A. H. Ross, P. Grob, M. A. Bothwell, D. E. Elder, C. S. Ernst, N. Marano, B. F. D. Ghrist, C. C. Slemp, M. Herlyn, B. Atkinson, and H. Koprowski, Proc. Natl. Acad. Sci. U.S.A. 81, 6681 (1984). 8 p. T. Kirschmeier, G. M. Housey, M. D. Johnson, A. S. Perkins, and I. B. Weinstein, D N A 7, 219 (1988). 9 M. Wigler, A. Pellicer, S. Silverstein, R. Axel, G. Urlaub, and L. Chasin, Proc. Natl. Acad. Sci. U.S.A. 76, 1373 (1979). 10 E. S. Schweitzer and R. B. Kelly, J. Cell Biol. 101, 667 (1985).

[6]

DETECTION OF N G F 5 LTR

RECEPTORS AFTER GENE TRANSFER

NGFR cONA

63

tk-Neomycin

NR18

I

G418 Selection

@ 1, Monoclonal anti-NGF receptor 2, RBC coated with Rabbit anti-Mouse IgG

FIG. 1. Detection of NGF receptors after CaPO4-mediated gene transfer. The recipient cell line is a PC12 variant which lacks endogenous receptors [M. A. Bothwell, A. L. Schechter, and K. M. Vaughn, Cell (Cambridge, Mass.) 21, 857 (1980)]. Receptors are detected after sequential incubation with anti-NGF receptor antibodies and red blood cells coupled to rabbit anti-mouse IgG. receptor. Transient transfection permits high level expression of cloned genes that can be a s s a y e d 12 to 80 hr later. In this case, plasmid sequences are maintained e x t r a c h r o m o s o m a l l y . F o r most cell lines, 1 - 1 0 % of the cells can give rise to significant expression.

Transfection 1. Plate cells at a density of 1 x l 0 6 cells/100 m m dish ! day before transfection. 2. Add dropwise 1.0 ml of CaPO 4 precipitate per dish. 9 Each milliliter of precipitate contains 2 0 / z g o f D N A , including high molecular weight carrier and plasmid D N A . 3. L e a v e precipitate on cells f r o m 6 to 12 hr in a CO 2 incubator. 4. Add 3 ml o f 20% glycerol in D M E (Dulbecco's modified Eagle's medium) for I min at r o o m temperature. 5. W a s h once with phosphate-buffered saline (PBS).

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6. Feed cells with nonselective medium. 7. Place into selective medium 1-2 days after transfection. Since expression of cell surface receptors is not amenable to metabolic selection, transfer of cloned genes depends on cotransfection with genes carrying selectable markers, most usually dominant acting genes. Neomycin (G418; GIBCO, Grand Island, NY) is the most widely used, and guanosine phosphoribosyltransferase (gpt), dihydrofolate reductase (dhfr), and hygromycin phosphotransferase (hygro) markers are also applicable. Resistance to drug selection will require 10-12 days in culture. For transient transfections, cells can be assayed (see below) 12-80 hr after transfection.

Retroviral Infection Low rates of gene transfer in cultured cells using conventional techniques can be overcome by retroviral infection with replication-defective vectors. Virus stocks are made by introduction of recombinant proviral DNA as plasmid DNA into packaging cells by using the standard calcium phosphate method outlined above (see Brown and Scott1'). Packaging lines contain a mutated helper virus that cannot replicate but can encode viral proteins required for production of encapsulated virions. Cultured supernatants produced from these packaging cells can be then used as a source of infectious retrovirus. The pMV7 vector is capable of generating neomycin-resistant colonies with a high probability of concomitant receptor expression, and it can also give rise to a stock of recombinant virus in packaging cell lines such as the amphotropic PA317 and the ecotropic tO2 line. These cells, transfected either stably or transiently with pMV7, can produce viral particles that can be used to infect susceptible target cells. The following procedure indicates the way in which infection with viral stocks is carried out. 1~ 1. Plate cells at 1 x 10 6 cells per 100 mm dish 1 day before infection. 2. Aspirate the medium and add viral supernatant [>103 plaque-forming units (pfu)/ml] and 8/~g/ml Polybrene (Aldrich, Milwaukee, WI) for 2 hr. 3. Add fresh medium for 1-2 days. 4. Place cells in selection. For most cells, G418 selection takes place at an optimum range between 0.6 and 0.8 mg/ml. Cell death will occur approximately 3 - 4 days after selection commences. II A. M. C. Brown and M. R. D. Scott, in " D N A Cloning, A Practical Approach" (D. M. Glover, ed.), Vol. 3, p. 189. IRL, Oxford, 1987.

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DETECTION OF N G F RECEPTORS AFTER GENE TRANSFER

65

FIG. 2. Positive rosette. Red blood cells coupled to affinity-purified goat anti-mouse IgG bind specifically to mouse fibroblasts expressing the human NGF receptor. A negative colony can be seen on the right side.

Erythrocyte Rosette Assay Cultured cells expressing specific cell surface antigens can be detected using monoclonal antibodies, and erythrocytes which are coupled to a second antibody. This binding can be visualized easily. Large numbers of colonies can be screened directly on a plate. Figure 1 shows the strategy for gene transfer and rosette assay for identification of cells expressing the human NGF receptor. The recipient cells (NR 18) are a derivative of PC 12 cells that are deficient in endogenous NGF receptors.12 The assay made use of a monoclonal antibody directed against the human melanoma NGF receptor, v The rosetting assay is a fast and sensitive technique for detecting the presence of cell surface proteins. Cultured cells expressing less than 1000 receptors per cell can be visualized. Figure 2 shows a strongly rosetting mouse Ltk ÷ colony expressing the human NGF receptor. Positives can be easily distinguished by the color of the coupled red blood cells (RBC) over an illuminated light source. Many tens of thousands of transformants 12 M. A. Bothwell, A. L. Schechter, and K. M. Vaughn, Cell (Cambridge, Mass.) 21, 857 (1980).

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can potentially be screened in only a few hours. When untransfected or receptor-negative cells are assayed in the same manner, no rosetting is observed. Since the monoclonal antibody recognizes only the human NGF receptor molecule, the expression of the NGF receptor in the transformants is not due to an activation of the endogenous NGF receptor.

Rosette Procedure 1. Wash plates 1-2 times with PBS (140 mM NaCI, 2.7 mM KCI, 8 mM NazHPO4, 1.5 mM KHzPO4). 2. Add antibody in PBS plus 5% fetal calf serum (FCS). (One must determine the appropriate titer on control cells. In general, ascites fluid is diluted at least 1 : 1000 and hybridoma supernatant 1 : 10.) Use 5 ml per plate. The ME20.4 hybridoma cell line is available from the ATCC (Rockville, MD). 3. Incubate for 1 hr at room temperature. 4. Wash 3 times with PBS. 5. Add 5-7 ml of coupled RBC. A 2% stock solution is diluted 1 : 10 in PBS plus 5% FCS (see below). 6. Incubate for 1 hr at room temperature. Positive rosettes can be seen after 1 hr, and positive colonies may be physically picked using cloning cylinders (Belco, Vineland, N J). The primary antibody can be reused.

Red Blood Cell Coupling The coupling of rabbit or goat anti-mouse IgG antibodies to human red blood cells is carried out according to the method of Goding '3 with several modifications. Human red cells are separated from their buffy coat using heparin, and chromic chloride is used to couple the antibody to red blood cells. The mixture is washed extensively in sterile saline and can be stored at 4 ° for up to 2 weeks in Hanks' balanced salt solution. 1. Draw human red blood cells in heparin tube. Spin for 5 min at room temperature (2000 rpm) to remove the buffy coats. 2. Wash the RBC 5-7 times in sterile saline (0.15 M NaCI) in a 50-ml tube. Do not use phosphate-buffered saline (PBS) buffer. 3. To 1 ml of packed RBC, add 2 ml of rabbit anti-mouse IgG antibody (Cappel, Malvern, PA, 0611-0082). Resuspend gently on vortex. The antibody has been previously dialyzed in saline at a concentration of 1 mg/ml. Affinity-purified antibodies are essential for specific rosette formation. 13 j. W. Goding, J. Immunol. Methods 10, 61 (1976).

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DETECTION OF N G F RECEPTORS AFTER GENE TRANSFER

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4. Add 2 ml working solution of CrC13 (C325; Fisher, Fairlawn, NJ) per milliliter of packed RBC, dropwisewhile the cells are constantly mixing (slow-speed vortex). The working solution is made from a 1 : 3 dilution of the stock solution (1 mg/ml in saline). The pH is adjusted to 5.0 with 1 M NaOH. 5. Rotate mixture at room temperature for 7 min. 6. Wash 5 times as in Step 2; however, now use cold (4°) saline. 7. Wash once in Hanks' balanced salt solution (HBSS; GIBCO). 8. Suspend 1 ml of packed RBC into 50 ml of HBSS to afinal concentration of 2%. This concentration is equivalent to a 10 × stock.

Immunofluorescence

Immunofluorescent analysis of transfected cells is easily carried out on glass coverslips. The cells can be directly exposed to a calcium phosphate precipitate of DNA on glass coverslips, or be replated after transfection. Indirect staining of human NGF receptors on transfected mouse fibroblasts is shown in Fig. 3. The cells were assayed transiently after gene transfer. The following procedure is modified after Rodriquez-Boulan.14 Plate cells on glass coverslips (Fisher) in 24-well plates. 2. Grow to 50-70% confluence. 3. Wash once with PBS. 4. Fix cells with 2% paraformaldehyde (in PBS) for 30 rain (or overnight at 4°). 5. Wash twice with PBS. 6. Quench with 50 mM NH4C1 in PBS for 20 min. 7. Wash twice with PBS plus 0.1% bovine serum albumin (BSA). 8. Incubate with the ME20.4 monoclonal antibody for 20 rain. 9. Wash 3 times with PBS/BSA solution. 10. Incubate with fluorescently tagged rabbit anti-mouse antibody (1 : 25 dilution) for 20 min. Antibodies are either F(ab')2 fragments or whole antibody coupled to fluorescein isothiocyanate (FITC) (Cappel). II. Wash 3 times with PBS/BSA solution. 12. Wash once with PBS. 13. Dip in water. 14. Mount on glass slides with a drop of Gelvatol (Monsanto Corp., St. Louis, MO). Gelvatol is polyvinyl alcohol (2.4 g in 6 ml glycerol, 6 ml H20, and 2 ml 0.2 M Tris, pH 8.5). .

14 E. Rodriguez-Boulan, this series, Vol. 98, p. 486.

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I G F , N G F , AND P D G F

[6]

FIG. 3. Indirect immunofluorescence of mouse fibroblasts transiently expressing human NGF receptors. (Top) Fluorescent cells following incubation with ME20.4 antibody 7 and FITC-labeled rabbit anti-mouse IgG. (Bottom) Phase-contrast micrograph of the same cells.

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DETECTION OF N G F RECEPTORS AFTER GENE TRANSFER

69

Affinity Labeling Affinity cross-linking of the receptor to [125I]NGF has become an effective and reproducible means of verifying the appearance of NGF receptor at the cell surface. Because NGF can be labeled to high specific activity by iodination, cell lines can be rapidly screened for NGF receptors. The most versatile cross-linking agent for detection of NGF receptors has been ethyldimethylaminopropylcarbodimide (EDAC), which reacts with carboxylic acid groups and primary amine groups. The major radioiodinated complex from the EDAC reaction of [125I]NGF bound to Schwann cells, neurons, pheochromocytoma, neuroblastoma, and melanoma cells has an apparent molecular weight of 100,000.15-17 Subtraction of the molecular weight of the fl subunit of NGF from the cross-linked complex gives an apparent molecular weight for the receptor of 75,000-85,000. The variation in the apparent molecular weight of the receptor is due to a considerable amount of glycosylation. A 200,000 MW protein found in most cross-linked receptor preparations has been shown to be a homodimer by peptide mapping.IS Equilibrium NGF binding experiments of mouse submaxillary [I~SI]NGF indicates that responsive neurons from the superior cervical or dorsal root ganglia and also cultured cells such as PC12 cells possess two different affinity forms, a high (Kd 10-11 M) and a low (K d l0 -9 M) affinity site. A second species (M r 135,000-160,000) can be identified by crosslinking [1zSI]NGF with the receptor using the heterofunctional and relatively lipophilic cross-linking agent hydroxysuccinimidyl 4-azidobenzoate (HSAB). This species probably represents an accessory protein that cooperates with the receptor to give rise to high affinity NGF binding. 5 For most studies, EDAC cross-linking is far more efficient and less difficult to achieve than cross-linking with HSAB. 4,17Disuccinimidyl suberate (DSS; Pierce, Rockford, IL), widely used in detecting other receptors, is also applicable to NGF receptor cross-linking. Since NGF is particularly unstable and binds to numerous surfaces and molecules, care must be taken in radiolabeling with iodine. Lactoperoxidase-catalyzed iodination is the most effective and nonevasive means of labeling NGF. The following protocol is an adaptation of the procedure I5 p. M. Grob, C. H. Berlot, and M. A. Bothwell, Proc. Natl. Acad. Sci. U.S.A. 80, 6819 (1985). 16 M. Taniuchi, E. M. Johnson, P. J. Roach, and J. C. Lawrence, J. Biol. Chem. 261, 13342 (1986). 17 S. H. Green and L. A. Greene, J. Biol. Chem. 261, 15316 (1986). 18 S. Buxser, P. Puma, and G. L. Johnson, J. Biol. Chem. 260, 1917 (1985).

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of Green and Greene. 17 An excellent description of current methods of purification of N G F can be found in Longo et al.19

Preparation of 125I-Labeled Nerve Growth Factor 1. Purified N G F or commercially obtained NGF (Bioproducts for Science, Indianapolis, IN) is aliquoted and kept at - 7 0 ° at a concentration of 1 mg/ml. 2. Add 10/xl of 1 mg/ml NGF to iodine lz5 (1 mCi/10/xl) from Amersham (IMS-30). 3. Add 6/xl of 50 ~g/ml lactoperoxidase (from ICN, Costa Mesa, CA). 4. Add 25/xl of 0.017% H202 (Sigma, St. Louis, MO), diluted in 0.2 M sodium phosphate, pH 6. 5. Incubate for 5 min at room temperature and vortex. Incubate for additional 5 min. 6. Quench the reaction with 50 ~1 of stop buffer [0.1 M NaI, 0.02% NaN 3, 0.1% BSA, 0.1% cytochrome c, 0.05% phenol red, 0.3% acetic acid (added before use)]. 7. Assess relative incorporation by trichloroacetic acid (TCA) precipitation of 2-~1 aliquots in 1 ml of 10% TCA. 8. Load the reaction on 0.7 x 25 cm P-100 (Bio-Rad, Richmond, CA) column equilibrated with 50 mM sodium phosphate, pH 7.4,100 mM NaC1, 0.5 mg/ml protamine sulfate (Sigma 4020), and 1 mg/ml bovine albumin (Sigma 7888). Aliquots of 0.2 ml volume are collected from the column. Labeled NGF appears at roughly twice the migratory distance as the phenol red indicator dye. A shoulder running ahead of the NGF peak represents a small amount of iodinated lactoperoxidase. The labeled NGF can be stored at 4 ° and must be used within 2 weeks.

Affinity Cross-Linking of Nerve Growth Factor Receptors 1. Wash cell monolayers twice with PBS. Cells are removed with PBS containing 1 mM EDTA, counted, and pelleted. 2. Resuspend cells in 1 ml of PBS, pH 6.5, to a final concentration of 2 - 4 × 10 6 cells/ml. 3. [~zSI]NGF is added to the cell suspension, and the mixture is rotated at 37° for 30 min. A typical concentration of NGF is 25 ng/ml; however, the concentration may vary according to experiment. As a control for the 19 F. M. Longo, J. E. Woo, and W. C. Mobley, in "Nerve Growth Factors" (R. A. Rush, ed.), p. 3. Wiley, New York, 1989.

[6]

DETECTION OF N G F RECEPTORS AFTER GENE TRANSFER

oO rr Z

~"

e40~

1"--

~

n," Z

0 n

71

~'~

"r

-200 kD

-103 - 67

FIG. 4. Affinity cross-linking analysis of the NGF receptor. [~25I]NGF was incubated with cultured cells, and the r e c e p t o r - N G F complex was cross-linked with the carbodiimide crosslinker EDAC. The detergent extract was subjected to polyacrylamide gel electrophoresis and autoradiography. Size markers are in kilodaltons.

specificity of the cross-linking, 2/zg of unlabeled NGF is incubated with an identical sample of cells with [125I]NGF. 4. EDAC (Pierce), made up in PBS, pH 6.5, is added to each sample to a final concentration of 4 mM, and the reaction allowed to proceed for 15 min at room temperature. 5. Cells are pelleted and washed once in PBS, pH 6.5. 6. Final pellets are resuspended in sodium dodecyl sulfate (SDS) loading buffer (plus 5% 2-mercaptoethanol), and the cross-linked products are resolved on 6 - 8 % polyacrylamide gels. The utility of the cross-linking reaction is demonstrated in Fig. 4. Four cell lines were incubated with 25 ng/ml [lZSI]NGF and cross-linked with EDAC. The "nonresponsive" NR18 line is a mutant line derived by mutagenesis of PC12 cells. J2 In agreement with previous observations, this cell line does not express any endogenous receptors similar to its parental PC12 line. A retrovirally transformed NR18 line called NRI-I has a small but detectable number of receptors: whereas human Hs294 melanoma and PC12 cells manifest many more receptors than the NRI-1 cell line. Each cell line displays a cross-linked product with the same apparent molecular size consistent with the size of the fully processed receptor

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conjugated with [125I]NGF. These cross-linking results indicate that the primary 75,000-85,000 dalton receptor species appears in cells with both high and low affinity forms of the receptor and supports the conclusion that the cloned NGF receptor gene is capable of giving rise to both high and low affinity receptors. 5 The heightened interest in the role of NGF in the central nervous system and in neurodegenerative diseases such as Alzheimer's dementia requires molecular and biochemical techniques to characterize the interaction of NGF and its receptor. The detection of NGF receptors by in situ rosetting, immunofluorescence, and affinity cross-linking provides the opportunity to use in vitro mutagenesis of NGF and its receptor to map the binding site and to delineate which structural features of the receptor are responsible for the functional responses of NGF.

[7] C l o n i n g a n d E x p r e s s i o n o f H u m a n P l a t e l e t - D e r i v e d G r o w t h F a c t o r a a n d fl R e c e p t o r s By LENA CLAESSON-WELSH, ANDERS ERIKSSON, BENGT WESTERMARK,

and CARL-HENgIK HELDIN Platelet-derived growth factor (PDGF), a major mitogen for connective tissue cells, is composed of disulfide-bonded A and B polypeptide chains. The three isoforms of PDGF have been found to bind to two distinct receptor types with different affinities (reviewed in Refs. 1 and 2). The PDGF/3 receptor (also denoted PDGF B-type receptor) binds PDGF-BB with high affinity, PDGF-AB with somewhat lower affinity, but appears not to bind PDGF-AA. The PDGF a receptor (also denoted PDGF Atype receptor) binds all PDGF isoforms with high affinity. Both receptors possess an intrinsic kinase activity, which becomes activated after ligand binding. Cloning and Expression of Human Platelet-Derived Growth Factor fl Receptor Partial amino acid sequence information of tryptic fragments from the purified murine PDGF/3 receptor was used to clone the corresponding C.-H. Heldin and B. Westermark, Trends Genet. 5, 108 (1989). z R. Ross, E. W. Raines, and D. F. Bowen-Pope,Cell (Cambridge, Mass.) 46, 155 (1986). METHODS IN ENZYMOLOGY, VOL. 198

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Detection of nerve growth factor receptors after gene transfer.

[6] DETECTION OF NGF RECEPTORS AFTER GENE TRANSFER 61 Acknowledgments I thank Michael S. Cole, Jeffrey K. Glenn, and Lori B. Taylor for technica...
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