Journal of Immunological Methods, 133 (1990) 107-118 Elsevier
107
JIM 05700
Characterization of polyclonal anti-peptide antibodies specific for transforming growth factor f12 A.J.M. Van den Eijnden-Van Raaij, I. Koornneef, H.G. Slager, C.L. M u m m e r y and E.J.J. Van Zoelen * Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
(Received 2 April 1990, accepted 7 June 1990)
An antiserum was prepared against a synthetic peptide corresponding to the first 29 N-terminal amino acid residues of transforming growth factor fl type 2 (TGFfl2) from porcine platelets. The anti-TGFfl2 peptide antiserum appeared to be completely specific for TGFfl2 in several immunological assays, including enzyme-linked immunosorbent assays, immunoblotting and immunofluorescence experiments. Furthermore, this antiserum completely neutralized the growth inhibitory effect of TGFfl2 on mink lung carcinoma (ML-CCI64) cells and the transforming capacity of this factor on quiescent monolayers of NRK cells in the presence of epidermal growth factor. These data indicate that the N-terminal region of TGFB2 may be involved in the biological activity of this growth factor. TGFfll was not recognized by the anti-TGFfl2 peptide antiserum. The specificity of the anti-TGFfl2 peptide antiserum for TGFfl2 appeared to be useful in identifying TGFfl2 produced by different cell systems and will be helpful in determining possible functional differences between TGFfll and TGFfl2. Key words: Transforming growth factor fl; Anti-peptide antiserum
Introduction
Transforming growth factor fl (TGFfl) is the prototype of a family of polypeptide factors that regulate cell growth and differentiation (Massagur, 1987). This highly ubiquitous molecule (Goustin et al., 1986) has been purified from several nonneoplastic tissues, transformed cells and from Correspondence to: A.J.M. Van den Eijnden-Van Raaij, Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. * Present address: Department of Cell Biology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. Abbreviations: TGFfl, transforming growth factor fl; EC, embryonal carcinoma cells; SDS, sodium dodecyl sulphate; ELISA, enzyme-linkedimmunosorbent assay.
media conditioned by several cell lines (Sporn et al., 1987). Intact TGFfl has a molecular weight of 25,000 and is composed of two identical disulfidelinked subunits of MW 12,500. It is now apparent that genetically distinct forms of TGFfl exist with similar structural features. The polypeptide originally described as TGFfl is now designated TGFfll (also known as cartilage-inducing factor A, CIF-A) and is the predominant species in human platelets. The second form of TGFfl, TGFfl2, is a homodimer of subunits which share 71% amino acid homology with TGFfll (Marquardt et al., 1987; Miller et al., 1989). This form has been isolated from bovine bone (known as cartilage-inducing factor B, CIFB) (Seyedin et al., 1985, 1987), from porcine platelets (Cheifetz et al., 1987) and also from media conditioned by human glioblastoma (Wrann
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
108 et al., 1987) and adenocarcinoma cells (Ikeda et al., 1987). Furthermore, the major growth inhibitory activity present in conditioned medium from African green monkey kidney epithelial cells (BSC-1 cells) has recently been identified as the homodimeric form of TGFfl2 (Hanks et al., 1988; McPherson et al., 1989). Recently, at least two additional types of TGFfl have been identified. TGFfl3 cDNAs were isolated from human (Derynck et al., 1988; Ten Dijke et al., 1988), porcine (Derynck et al., 1988) and chicken (Jakowlew et al., 1988a) cDNA libraries while TGFfl4 cDNAs were obtained from a chicken chondrocyte library (Jakowlew et al., 1988b). In agreement with TGFfll and TGFfl2 these cDNA sequences predict synthesis of both TGFfl3 and TGFfl4 as precursor polypeptides from which the mature part is derived by dibasic cleavage. There is no signal peptide sequence in the precursor TGFfl4 protein. The biological properties of mature TGFfl3 and TGFfl4 have not been determined as yet due to the lack of sufficient purified material. In most mammalian cell systems tested sofar the biological activities of TGFflI and TGFfl2 have been found to be indistinguishable (Seyedin et al., 1985, 1987; Cheifetz et al., 1987). However, a haemopoietic cell line has recently been shown to be growth-inhibited by TGFfl1 but not by TGFfl2 (Ohta et al., 1987) although little or no difference between the two factors was observed by Keller et al. (1989). Furthermore, in the amphibian embryo TGFfl2 itself, but not TGFfl1, is active in mesoderm induction (Rosa et al., 1988). In order to distinguish between the functional role of TGFfl1 and TGFfl2 in biological systems specific neutralizing anti-TGFfl antibodies are needed and a number of different antibodies have recently been raised against peptide determinants of TGFfl1, purified TGFfll, or purified TGFfl2 (Ellingsworth et al., 1986; Keski-Oja et al., 1987; Flanders et al., 1988; Rosa et al., 1988; Danielpour et al., 1989). From these studies it seemed to be very difficult to generate antibodies against the native proteins that are specific for each of the two forms, due to the high degree of amino acid homology between them. Furthermore, large amounts of pure growth factor are required for immunization. In the present study we have raised antibodies against a
synthetic oligopeptide identical to the N-terminal residues 1-29 of TGFfl2. This region shows the least homology between TGFfll and TGFfl2 with 11 amino acid differences in the first 29 amino acids of the mature factors. This approach has resulted in polyclonal antibodies that specifically recognize TGFfl2 in a variety of immunological assays and completely neutralize the effects of TGFfl2 in several bioassays. To our knowledge this is the first report of TGFflE-specific neutralizing anti-peptide antibodies. These antibodies should be useful for the detection and localization of TGFfl2 protein in cells and tissues by immunohistochemical studies, and in determining the function of this growth factor by in vitro and in vivo studies.
Materials and methods
TGFfl Human TGFfl1 was purified from outdated platelets according to the method of Van den Eijnden-Van Raaij et al. (1988). Purified TGFfl2 from porcine platelets was obtained from R and D Systems (Minneapolis). P olypeptide synthesis Polypeptides corresponding to the N-terminal amino acid sequences 1-29 of TGFfll (peptide B1) and of TGFfl2 (peptide B2) were synthesized on a SamlI peptide synthesizer (Biosearch, New Brunschwig Scientific) using t-Boc protection methodology (Barany et al., 1980). The peptides were assembled on p-methylbenzhydrylamine resin, cleaved from the solid phase resin and deprotected with a mixture of trifluoromethane sulfonic acid, thioanisole and trifluoroacetic acid. The amino acid composition was verified using the Pico-Tag amino acid analysis system (Waters). Production of antibodies against TGFfl New Zealand White rabbits were immunized by intramuscular injections of the uncoupled peptides biweekly for 8 weeks using 250 #g of peptide per injection. The primary immunization was in Freund's complete adjuvant and the subsequent boosts were in incomplete Freund' s adjuvant. The sera were collected from blood drawn 2 weeks
109 following the final boost. For the production of antibodies against native human platelet TGFfl1, rabbits were immunized at intramuscular sites with four doses of keyhole limpet haemocyanin (KLH)-coupled TGFfl (100 gg of TGFfl per dose per rabbit) in Freund's adjuvant at 3 week intervals. The rabbits were bled 5 weeks after boosting.
allowed to develop with the peroxidase substrate. The substrate consisted of 0.2% (w/v) o-phenylenediamine (OPD) and 0.015% (v/v) H202 in a 25 mM citrate buffer (pH 5.0). After incubating for 10 min at room temperature in the dark the reaction was stopped by adding 50 gl 2 M sulphuric acid to the wells. The optical density was determined at 492 nm using a Titertek Multiscan.
Purification of antisera IgG fractions of the different antisera were purified by affinity chromatography on protein A-Sepharose (Goeding, 1978). The IgG fraction of normal rabbit preimmune serum served as a control. The IgG fractions were extensively dialysed against phosphate-buffered saline (PBS) and concentrated in a collodion bag. The anti-peptide antisera were also affinity-purified by incubation with a column of Reactigel HW-65F (agarose gel beads 0.5 ml; Pierce) that had been coupled to 1 mg of the appropriate peptide according to the manufacturer's instructions (Gullick et al., 1985). The flowthrough from the column was tested for its ability to recognize TGFfl peptide in an ELISA assay (below). The agarose column was washed with PBS and specifically bound antibodies were eluted with 50 mM glycine-HC1, pH 2.0. The eluate was immediately neutralized with 1 M Na2HPO 4, dialysed against PBS and concentrated in a collodion bag.
ELISA The various antisera were tested for their reactivity with the different antigens by an ELISA method. The antigens were solubilized in 66% acetonitrile/10 mM trifluoroacetic acid and diluted in 50 mM carbonate buffer (pH 9.8). The wells of a microtitre plate were coated with antigen solution overnight at 37°C. The plates were air-dried and non-specific binding was blocked with DMEM-Hepes medium containing 1% ovalbumin (blocking buffer) for 1 h at 37°C. The antisera to be tested were added to the wells in blocking buffer at a dilution of 1/100 and incubated for 1 h at 37°C. The plates were washed with PBS-buffered 0.05% (v/v) Tween 20, and peroxidase-conjugated goat anti-rabbit IgG was added for 1 h at 37°C (1/1000 diluted in blocking buffer). The plates were washed extensively with PBS containing 0.05% Tween 20 and color was
SDS-polyacrylamide gel electrophoresis Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970) using a 15% polyacrylamide separating gel and a 5% stacking gel. Protein samples were solubilized in 5% SDS in the absence of reducing agents by heating at 100°C for 5 min. The gels were electrophoretically transferred to nitrocellulose (below).
Immunoblotting Gels were blotted overnight on to nitrocellulose paper according to the method of Towbin et al. (1979). The nitrocellulose papers containing the protein antigens were incubated with the primary antibody (1/50 dilution) for 1 h at 37°C. The immune complexes were visualized as described by Van Zoelen et al. (1985).
Immunoprecipitation of radiolabelled cell media BSC-1 cells (ATCC, CCL26) were grown to confluency in Dulbecco's modified Eagle's medium (DMEM) containing 7.5% fetal calf serum. Prior to labelling the cells were washed twice with serum-free Eagle's minimal essential medium and once with cysteine-free medium. Afterwards the cells were labelled with L-[3SS]cysteine (100 gCi/ml, 1300 gCi/mmol) in the cysteine-free medium for 6 h. 10 ml medium were collected, clarified by centrifugation and treated at pH 1.5 for 1 h, followed by neutralization. Parallel samples were not acidified thus leaving TGFfl in the latent form. The media were then subjected to immunoprecipitation analysis. The samples were preprecipitated with normal rabbit serum and protein A-Sepharose and then incubated overnight at 4°C with a 1/50 dilution of test antibody, followed by precipitation with protein A-Sepharose. The specifity of the immunoprecipitation was determined by preincubating the antibody with 120
110
ng cold TGFfl2. The bound material was eluted by boiling in Laemmli's sample buffer and electrophoresed in a non-reducing 15% SDS-polyacrylamide gel. After electrophoresis the gels were treated with Amplify (Amersham), dried and exposed to Kodak at - 7 0 ° C for 2 weeks.
Inhibition of TGFfl receptor binding by anti-TGFfl antisera TGFfll was radioiodinated by chloramine-T according to the method of Frolik et al. (1984). 6 ng/ml 125I-TGFfll (27 gCi/gg) in binding buffer (MEM, containing 0.1% BSA, 25 mM Hepes, pH 7.4) were preincubated at room temperature with various dilutions of test antibodies and then added to subconfluent monolayers of NRK-49F cells. After incubation at room temperature for 3 h the cells were washed with binding buffer and bound radioactivity was extracted with lysis buffer (Frolik et al., 1984).
Inhibition of TGFfl biological activity the anti-TGFfl antisera TGFfll or TGFfl2 were preincubated for 1 h at 37°C with appropriate dilutions of the various antisera or control serum in DMEM containing 0.1% BSA. Subsequently triplicate aliquots were tested for the presence of TGFfl activity by their growth inhibitory action on exponentially growing mink lung CC164 cells resulting in a reduction of [3H]thymidine incorporation (Van Zoelen et al., 1987a) as modified by Rosa et al. (1988). In addition, the presence of TGFfl activity was determined by the loss of density-dependent inhibition of growth of N R K cells. [3H]tliymidine uptake was measured in the presence of EGF (5 ng/ml) and insulin (5 gg/ml) during a 2 h time pulse between 43 and 45 h after mitogenic stimulation of confluent serum-free cultures (Van Zoelen et al., 1987b).
(END-2) derived from P19 EC cells (Mummery et al., 1985) and the TGFflE-producing African green monkey kidney epithelial cell line BSC-1. Except for the BSC-1 cell line the cells were grown on gelatin-coated glass coverslips to 60-70% confluency in a 1 : 1 mixture of Dulbecc.o's modified Eagle's medium (DMEM) and Ham F12 medium containing 44 mM NaHCO 3 (pH 7.6; DF-BIC medium) supplemented with 7.5% fetal calf serum (FCS, Gibco). Subsequently the cells were incubated in serum-free medium, fixed with 2% glutaraldehyde in PBS for 20 rain at room temperature (RT), quenched with 0.5 mg/ml sodium borohydride in PBS for 5 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 4 min at room temperature. Afterwards the cells were first preincubated with PBS containing 2 m g / m l ovalbumin for 1 h at room temperature and then exposed to affinity-purified antiTGFfl antisera or control serum (0.08 # g / g l in PBS + 2 m g / m l ovalbumin) for 1 h at RT, rinsed with PBS, incubated with fluorescein isothiocyanate conjugated goat anti-rabbit IgG (diluted 1/40 in PBS/ovalbumin) for 1 h at RT, rinsed with PBS and mounted in Moviol.
Results
Preparation and ELISA reactivity of anti-TGFfl2 antibodies A peptide corresponding to the first 29 Nterminal amino acid residues of human TGFfl2 (peptide B2) was synthesized and used to immunize rabbits without prior coupling to a carder protein. For comparison, the corresponding TGFfll peptide (peptide B1) (see also Ellingsworth et al., 1986) was used for immunization. As shown in Fig. 1 there are 11 amino acid differences between these two peptides. The substantial sequence di-
TGFflI
Indirect immunofluorescence The presence of TGFfl1 and TGFfl2 in different cell types was determined by indirect immunofluorescence using the anti-peptide antisera. The cell systems studied were P19 embryonal carcinoma (EC) cells, before and after treatment with the differentiation-inducing agent retinoic acid (RA), a differentiated endodermal clone
NP,2~ l
TGF~2 " ~
K P
Fig. L A~l~noacid s~uenc¢of the N-ten-nina]residues1-29 of TGF~I (pepfide B1) and TGFp2 (peptide B2).
111
2.0
c ¢ peptide B1 o ~ o peptide B2
k
\
*- -"TGF81
¢q O~
107
JIM 05700
Characterization of polyclonal anti-peptide antibodies specific for transforming growth factor f12 A.J.M. Van den Eijnden-Van Raaij, I. Koornneef, H.G. Slager, C.L. M u m m e r y and E.J.J. Van Zoelen * Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
(Received 2 April 1990, accepted 7 June 1990)
An antiserum was prepared against a synthetic peptide corresponding to the first 29 N-terminal amino acid residues of transforming growth factor fl type 2 (TGFfl2) from porcine platelets. The anti-TGFfl2 peptide antiserum appeared to be completely specific for TGFfl2 in several immunological assays, including enzyme-linked immunosorbent assays, immunoblotting and immunofluorescence experiments. Furthermore, this antiserum completely neutralized the growth inhibitory effect of TGFfl2 on mink lung carcinoma (ML-CCI64) cells and the transforming capacity of this factor on quiescent monolayers of NRK cells in the presence of epidermal growth factor. These data indicate that the N-terminal region of TGFB2 may be involved in the biological activity of this growth factor. TGFfll was not recognized by the anti-TGFfl2 peptide antiserum. The specificity of the anti-TGFfl2 peptide antiserum for TGFfl2 appeared to be useful in identifying TGFfl2 produced by different cell systems and will be helpful in determining possible functional differences between TGFfll and TGFfl2. Key words: Transforming growth factor fl; Anti-peptide antiserum
Introduction
Transforming growth factor fl (TGFfl) is the prototype of a family of polypeptide factors that regulate cell growth and differentiation (Massagur, 1987). This highly ubiquitous molecule (Goustin et al., 1986) has been purified from several nonneoplastic tissues, transformed cells and from Correspondence to: A.J.M. Van den Eijnden-Van Raaij, Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. * Present address: Department of Cell Biology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. Abbreviations: TGFfl, transforming growth factor fl; EC, embryonal carcinoma cells; SDS, sodium dodecyl sulphate; ELISA, enzyme-linkedimmunosorbent assay.
media conditioned by several cell lines (Sporn et al., 1987). Intact TGFfl has a molecular weight of 25,000 and is composed of two identical disulfidelinked subunits of MW 12,500. It is now apparent that genetically distinct forms of TGFfl exist with similar structural features. The polypeptide originally described as TGFfl is now designated TGFfll (also known as cartilage-inducing factor A, CIF-A) and is the predominant species in human platelets. The second form of TGFfl, TGFfl2, is a homodimer of subunits which share 71% amino acid homology with TGFfll (Marquardt et al., 1987; Miller et al., 1989). This form has been isolated from bovine bone (known as cartilage-inducing factor B, CIFB) (Seyedin et al., 1985, 1987), from porcine platelets (Cheifetz et al., 1987) and also from media conditioned by human glioblastoma (Wrann
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
108 et al., 1987) and adenocarcinoma cells (Ikeda et al., 1987). Furthermore, the major growth inhibitory activity present in conditioned medium from African green monkey kidney epithelial cells (BSC-1 cells) has recently been identified as the homodimeric form of TGFfl2 (Hanks et al., 1988; McPherson et al., 1989). Recently, at least two additional types of TGFfl have been identified. TGFfl3 cDNAs were isolated from human (Derynck et al., 1988; Ten Dijke et al., 1988), porcine (Derynck et al., 1988) and chicken (Jakowlew et al., 1988a) cDNA libraries while TGFfl4 cDNAs were obtained from a chicken chondrocyte library (Jakowlew et al., 1988b). In agreement with TGFfll and TGFfl2 these cDNA sequences predict synthesis of both TGFfl3 and TGFfl4 as precursor polypeptides from which the mature part is derived by dibasic cleavage. There is no signal peptide sequence in the precursor TGFfl4 protein. The biological properties of mature TGFfl3 and TGFfl4 have not been determined as yet due to the lack of sufficient purified material. In most mammalian cell systems tested sofar the biological activities of TGFflI and TGFfl2 have been found to be indistinguishable (Seyedin et al., 1985, 1987; Cheifetz et al., 1987). However, a haemopoietic cell line has recently been shown to be growth-inhibited by TGFfl1 but not by TGFfl2 (Ohta et al., 1987) although little or no difference between the two factors was observed by Keller et al. (1989). Furthermore, in the amphibian embryo TGFfl2 itself, but not TGFfl1, is active in mesoderm induction (Rosa et al., 1988). In order to distinguish between the functional role of TGFfl1 and TGFfl2 in biological systems specific neutralizing anti-TGFfl antibodies are needed and a number of different antibodies have recently been raised against peptide determinants of TGFfl1, purified TGFfll, or purified TGFfl2 (Ellingsworth et al., 1986; Keski-Oja et al., 1987; Flanders et al., 1988; Rosa et al., 1988; Danielpour et al., 1989). From these studies it seemed to be very difficult to generate antibodies against the native proteins that are specific for each of the two forms, due to the high degree of amino acid homology between them. Furthermore, large amounts of pure growth factor are required for immunization. In the present study we have raised antibodies against a
synthetic oligopeptide identical to the N-terminal residues 1-29 of TGFfl2. This region shows the least homology between TGFfll and TGFfl2 with 11 amino acid differences in the first 29 amino acids of the mature factors. This approach has resulted in polyclonal antibodies that specifically recognize TGFfl2 in a variety of immunological assays and completely neutralize the effects of TGFfl2 in several bioassays. To our knowledge this is the first report of TGFflE-specific neutralizing anti-peptide antibodies. These antibodies should be useful for the detection and localization of TGFfl2 protein in cells and tissues by immunohistochemical studies, and in determining the function of this growth factor by in vitro and in vivo studies.
Materials and methods
TGFfl Human TGFfl1 was purified from outdated platelets according to the method of Van den Eijnden-Van Raaij et al. (1988). Purified TGFfl2 from porcine platelets was obtained from R and D Systems (Minneapolis). P olypeptide synthesis Polypeptides corresponding to the N-terminal amino acid sequences 1-29 of TGFfll (peptide B1) and of TGFfl2 (peptide B2) were synthesized on a SamlI peptide synthesizer (Biosearch, New Brunschwig Scientific) using t-Boc protection methodology (Barany et al., 1980). The peptides were assembled on p-methylbenzhydrylamine resin, cleaved from the solid phase resin and deprotected with a mixture of trifluoromethane sulfonic acid, thioanisole and trifluoroacetic acid. The amino acid composition was verified using the Pico-Tag amino acid analysis system (Waters). Production of antibodies against TGFfl New Zealand White rabbits were immunized by intramuscular injections of the uncoupled peptides biweekly for 8 weeks using 250 #g of peptide per injection. The primary immunization was in Freund's complete adjuvant and the subsequent boosts were in incomplete Freund' s adjuvant. The sera were collected from blood drawn 2 weeks
109 following the final boost. For the production of antibodies against native human platelet TGFfl1, rabbits were immunized at intramuscular sites with four doses of keyhole limpet haemocyanin (KLH)-coupled TGFfl (100 gg of TGFfl per dose per rabbit) in Freund's adjuvant at 3 week intervals. The rabbits were bled 5 weeks after boosting.
allowed to develop with the peroxidase substrate. The substrate consisted of 0.2% (w/v) o-phenylenediamine (OPD) and 0.015% (v/v) H202 in a 25 mM citrate buffer (pH 5.0). After incubating for 10 min at room temperature in the dark the reaction was stopped by adding 50 gl 2 M sulphuric acid to the wells. The optical density was determined at 492 nm using a Titertek Multiscan.
Purification of antisera IgG fractions of the different antisera were purified by affinity chromatography on protein A-Sepharose (Goeding, 1978). The IgG fraction of normal rabbit preimmune serum served as a control. The IgG fractions were extensively dialysed against phosphate-buffered saline (PBS) and concentrated in a collodion bag. The anti-peptide antisera were also affinity-purified by incubation with a column of Reactigel HW-65F (agarose gel beads 0.5 ml; Pierce) that had been coupled to 1 mg of the appropriate peptide according to the manufacturer's instructions (Gullick et al., 1985). The flowthrough from the column was tested for its ability to recognize TGFfl peptide in an ELISA assay (below). The agarose column was washed with PBS and specifically bound antibodies were eluted with 50 mM glycine-HC1, pH 2.0. The eluate was immediately neutralized with 1 M Na2HPO 4, dialysed against PBS and concentrated in a collodion bag.
ELISA The various antisera were tested for their reactivity with the different antigens by an ELISA method. The antigens were solubilized in 66% acetonitrile/10 mM trifluoroacetic acid and diluted in 50 mM carbonate buffer (pH 9.8). The wells of a microtitre plate were coated with antigen solution overnight at 37°C. The plates were air-dried and non-specific binding was blocked with DMEM-Hepes medium containing 1% ovalbumin (blocking buffer) for 1 h at 37°C. The antisera to be tested were added to the wells in blocking buffer at a dilution of 1/100 and incubated for 1 h at 37°C. The plates were washed with PBS-buffered 0.05% (v/v) Tween 20, and peroxidase-conjugated goat anti-rabbit IgG was added for 1 h at 37°C (1/1000 diluted in blocking buffer). The plates were washed extensively with PBS containing 0.05% Tween 20 and color was
SDS-polyacrylamide gel electrophoresis Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970) using a 15% polyacrylamide separating gel and a 5% stacking gel. Protein samples were solubilized in 5% SDS in the absence of reducing agents by heating at 100°C for 5 min. The gels were electrophoretically transferred to nitrocellulose (below).
Immunoblotting Gels were blotted overnight on to nitrocellulose paper according to the method of Towbin et al. (1979). The nitrocellulose papers containing the protein antigens were incubated with the primary antibody (1/50 dilution) for 1 h at 37°C. The immune complexes were visualized as described by Van Zoelen et al. (1985).
Immunoprecipitation of radiolabelled cell media BSC-1 cells (ATCC, CCL26) were grown to confluency in Dulbecco's modified Eagle's medium (DMEM) containing 7.5% fetal calf serum. Prior to labelling the cells were washed twice with serum-free Eagle's minimal essential medium and once with cysteine-free medium. Afterwards the cells were labelled with L-[3SS]cysteine (100 gCi/ml, 1300 gCi/mmol) in the cysteine-free medium for 6 h. 10 ml medium were collected, clarified by centrifugation and treated at pH 1.5 for 1 h, followed by neutralization. Parallel samples were not acidified thus leaving TGFfl in the latent form. The media were then subjected to immunoprecipitation analysis. The samples were preprecipitated with normal rabbit serum and protein A-Sepharose and then incubated overnight at 4°C with a 1/50 dilution of test antibody, followed by precipitation with protein A-Sepharose. The specifity of the immunoprecipitation was determined by preincubating the antibody with 120
110
ng cold TGFfl2. The bound material was eluted by boiling in Laemmli's sample buffer and electrophoresed in a non-reducing 15% SDS-polyacrylamide gel. After electrophoresis the gels were treated with Amplify (Amersham), dried and exposed to Kodak at - 7 0 ° C for 2 weeks.
Inhibition of TGFfl receptor binding by anti-TGFfl antisera TGFfll was radioiodinated by chloramine-T according to the method of Frolik et al. (1984). 6 ng/ml 125I-TGFfll (27 gCi/gg) in binding buffer (MEM, containing 0.1% BSA, 25 mM Hepes, pH 7.4) were preincubated at room temperature with various dilutions of test antibodies and then added to subconfluent monolayers of NRK-49F cells. After incubation at room temperature for 3 h the cells were washed with binding buffer and bound radioactivity was extracted with lysis buffer (Frolik et al., 1984).
Inhibition of TGFfl biological activity the anti-TGFfl antisera TGFfll or TGFfl2 were preincubated for 1 h at 37°C with appropriate dilutions of the various antisera or control serum in DMEM containing 0.1% BSA. Subsequently triplicate aliquots were tested for the presence of TGFfl activity by their growth inhibitory action on exponentially growing mink lung CC164 cells resulting in a reduction of [3H]thymidine incorporation (Van Zoelen et al., 1987a) as modified by Rosa et al. (1988). In addition, the presence of TGFfl activity was determined by the loss of density-dependent inhibition of growth of N R K cells. [3H]tliymidine uptake was measured in the presence of EGF (5 ng/ml) and insulin (5 gg/ml) during a 2 h time pulse between 43 and 45 h after mitogenic stimulation of confluent serum-free cultures (Van Zoelen et al., 1987b).
(END-2) derived from P19 EC cells (Mummery et al., 1985) and the TGFflE-producing African green monkey kidney epithelial cell line BSC-1. Except for the BSC-1 cell line the cells were grown on gelatin-coated glass coverslips to 60-70% confluency in a 1 : 1 mixture of Dulbecc.o's modified Eagle's medium (DMEM) and Ham F12 medium containing 44 mM NaHCO 3 (pH 7.6; DF-BIC medium) supplemented with 7.5% fetal calf serum (FCS, Gibco). Subsequently the cells were incubated in serum-free medium, fixed with 2% glutaraldehyde in PBS for 20 rain at room temperature (RT), quenched with 0.5 mg/ml sodium borohydride in PBS for 5 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 4 min at room temperature. Afterwards the cells were first preincubated with PBS containing 2 m g / m l ovalbumin for 1 h at room temperature and then exposed to affinity-purified antiTGFfl antisera or control serum (0.08 # g / g l in PBS + 2 m g / m l ovalbumin) for 1 h at RT, rinsed with PBS, incubated with fluorescein isothiocyanate conjugated goat anti-rabbit IgG (diluted 1/40 in PBS/ovalbumin) for 1 h at RT, rinsed with PBS and mounted in Moviol.
Results
Preparation and ELISA reactivity of anti-TGFfl2 antibodies A peptide corresponding to the first 29 Nterminal amino acid residues of human TGFfl2 (peptide B2) was synthesized and used to immunize rabbits without prior coupling to a carder protein. For comparison, the corresponding TGFfll peptide (peptide B1) (see also Ellingsworth et al., 1986) was used for immunization. As shown in Fig. 1 there are 11 amino acid differences between these two peptides. The substantial sequence di-
TGFflI
Indirect immunofluorescence The presence of TGFfl1 and TGFfl2 in different cell types was determined by indirect immunofluorescence using the anti-peptide antisera. The cell systems studied were P19 embryonal carcinoma (EC) cells, before and after treatment with the differentiation-inducing agent retinoic acid (RA), a differentiated endodermal clone
NP,2~ l
TGF~2 " ~
K P
Fig. L A~l~noacid s~uenc¢of the N-ten-nina]residues1-29 of TGF~I (pepfide B1) and TGFp2 (peptide B2).
111
2.0
c ¢ peptide B1 o ~ o peptide B2
k
\
*- -"TGF81
¢q O~
