From the Departments of H-kininogen is a multifunctional protein: it
inhibits cysteine proteases, plays a role in contact activation of the
coagulation cascade, and is the precursor of the potent proinflammatory
peptide bradykinin. In the experiments described here, we identify
H-kininogen as a ferritin-binding protein. Ferritin is a cellular and
serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and
used it to guide purification of a ferritin-binding protein from human
serum. Edman degradation of the purified protein determined the
sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues
421-436 of human H-kininogen. These results were confirmed by
demonstrating that commercially purified H-kininogen possessed ferritin
binding activity and that ferritin binding could not be detected in
plasma from kininogen-deficient individuals. Ligand blot assays mapped
the ferritin binding domain to the light chain of H-kininogen chain,
and revealed that both H and L recombinant ferritins possess
H-kininogen binding activity. The unexpected identification of
H-kininogen as a ferritin-binding protein may link ferritin in the
complex chain of interactions by which H-kininogen mediates its
multiple effects in contact activation and inflammation.
Ferritin is an iron-binding protein that is found in most human
tissues and in human serum. It is a 24-subunit protein composed of two
chains, termed H and L (see Refs. 1-3 for review). Tissue ferritins
have been extensively characterized, but serum ferritins much less so.
Previous studies have shown that in addition to ferritin, human serum
contains ferritin-binding protein(s) (4-6). In one study, partial
purification revealed ferritin binding activity to be present in the
H-kininogen is a 626-amino acid plasma glycoprotein with pleiotrophic
functions (reviewed in Ref. 15). Residues 363-371 of H-kininogen
encode bradykinin, a potent vasoactive nonapeptide that is released
from H-kininogen through a succession of proteolytic reactions mediated
by plasma kallikrein (16). Effects of bradykinin include stimulation of
prostaglandin and leukotriene production, tissue type plasminogen
activator release, nitric oxide release, contraction of vascular smooth
muscle cells, and pain (15). As a plasma protein, H-kininogen plays a
role in contact phase activation of the coagulation cascade and
functions as a cysteine protease inhibitor. In addition to its presence
in plasma, H-kininogen has also been localized on the surface of a
variety of cells, including endothelial cells (17), platelets (18), and
neutrophils (19). H-kininogen maintains its ability to bind kallikrein
when docked at the surface of these cells, and it has been proposed that the localized release of bradykinin that occurs at these sites
contributes to diapedesis or localized contact activation (19).
Ferritin is elevated during acute and chronic inflammation, and the
observation that ferritin and H-kininogen interact suggests that
ferritin may modulate some of the effects of H-kininogen, perhaps
during the inflammatory response.
Ligand Blotting--
Proteins were electrophoresed in 7%
SDS-PAGE1 gels (20). Samples
were prepared by heating at 90 °C for 5 min in sample buffer containing 1% SDS, 30 mM Tris, pH 6.8, 5% glycerol, and
no reducing agents. Following electrophoresis, proteins were
transferred to a nitrocellulose membrane overnight at 4 °C in 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3. Following transfer, the membrane was blocked with 5% nonfat dry milk
in phosphate-buffered saline (PBS) for 1 h at room temperature,
washed in PBS, incubated with 4 µg/ml ferritin from human spleen
(>98% pure; Scripps Laboratories) in 1% bovine serum albumin (Sigma)
in PBS for 2 h at room temperature, washed three times in PBS, and
incubated with horseradish peroxidase-conjugated antiferritin antibody
(Dako) at a 1:5000 dilution for 1 h at room temperature. In some
experiments, blots were incubated with recombinant human ferritin H or
recombinant human ferritin L purified as described (21). In some cases,
blots were incubated with a monoclonal antiferritin antibody
(Biospecific) followed by HRP-conjugated anti-mouse antibody. Bound
antibody was revealed using a chemiluminescent substrate (ECL, Amersham
Pharmacia Biotech), followed by exposure for several seconds to x-ray
film. When signal intensities were very strong, the blot was stored for
1 h to allow the signal to decay prior to exposing the film. Band
intensity was quantitated by scanning densitometry (PDI325, Amersham
Pharmacia Biotech).
Ferritin-binding Protein Purification--
Blood was collected
from normal volunteers and allowed to coagulate at room temperature for
15 min. Crude serum was obtained by centrifugation for 10 min at
4 °C. EDTA (pH 8.0) and phenylmethylsulfonyl fluoride (Sigma) were
added to a final concentration of 0.5 mM and 0.2 µM, respectively, followed by solid ammonium sulfate to achieve 50% saturation. After 2 h, the precipitate was collected by centrifugation at 23,000 × g for 30 min, dissolved
in cold 100 mM sodium acetate/0.1 M NaCl, pH
5.8, and dialyzed overnight at 4 °C against the same buffer. The
solution was clarified by centrifugation and applied to a CM-Sephadex
C-50 Column (Amersham Pharmacia Biotech) equilibrated with 0.1 M sodium acetate/0.1 M NaCl, pH 5.8. After
washing the column with 10 ml of the starting buffer, elution was
accomplished by initiating a linear gradient of 0.1-0.7 M
NaCl in 100 mM sodium acetate buffer, pH 5.8. Fractions of
2 ml were collected at a flow rate of 20 ml/h and assayed for protein
concentration and ferritin binding activity. Active fractions were
pooled, lyophilized, resuspended in cold water, and dialyzed extensively at 4 °C against PBS containing 0.2 µM
phenylmethylsulfonyl fluoride. Protein concentration was measured using
either the Bio-Rad or the Quantigold (Diversified Biotech) assay, with
bovine serum albumin as standard. For amino acid sequencing,
approximately 10 µg of partially purified protein was electrophoresed
on a 7% SDS-PAGE gel, transferred to a polyvinylidene difluoride
(PVDF) membrane (Stratagene), and stained with Coomassie Blue. The
~100-kDa region of the gel was excised and N-terminal sequence
analysis performed by Edman degradation. Data base comparisons were
performed using the BLAST program (Genetics Computer Group, Madison,
WI).
Western Blot Detection of H-kininogen--
Purified H-kininogen
and L-kininogen were obtained from Calbiochem and were detected on
Western blots using HRP-conjugated antikininogen antibody (The Binding
Site Ltd.). Kininogen-deficient plasma was obtained from an individual
with congenital kininogen deficiency (Fitzgerald trait) and was
supplied by George King Biomedical, Inc. Normal reference plasma was
obtained from Precision Biologicals (Dartmouth, Nova Scotia,
Canada).
Gel Electrophoresis--
Native gels were prepared by omitting
SDS from all reagents used in the preparation of a Laemmli gel (20),
including sample buffer.
Identification of Ferritin-binding Proteins by Ligand
Blotting--
In order to determine whether a ferritin binding
activity could be identified in human serum, we performed a ligand blot
assay. This general method was originally described by Brown and
Goldstein and their co-workers (22) in their analysis of the LDL
receptor and has since been used to identify ligand/receptor
interactions for a number of diverse ligands, including tumor necrosis
factor (23) and insulin-like growth factor-1 (24). Human serum was dissolved in SDS-PAGE sample buffer without reducing agents, subjected to SDS-PAGE, and transferred to nitrocellulose. The blot was incubated with ferritin, followed by HRP-conjugated antiferritin antibody and a
chemiluminescent substrate. Ferritin-binding proteins were thus
detected by the formation of a ferritin-binding
protein-ferritin-antibody complex on the blot. As shown in Fig.
1A, this assay detected three
specific bands. As also illustrated in Fig. 1A, controls demonstrated that reactivity in this assay was not observed if either
ferritin or anti-ferritin antibody was omitted (lanes 6 and
7, respectively). Furthermore, a monoclonal anti-ferritin antibody, as well as a polyclonal anti-ferritin antibody, was able to
detect ferritin-binding proteins (lane 1); however, these bands were not detected if an unrelated antibody (anti-rabbit IgG) was
substituted for antiferritin antibody (not shown). Incubation with
ferritin did not nonspecifically enhance the binding of antibodies to
the blot; for example, it did not affect the reactivity of the blot to
anti-rabbit IgG (not shown).
Biochemistry,
Internal Medicine, Wake Forest University School of Medicine
and the Comprehensive Cancer Center of Wake Forest University,
Winston-Salem, North Carolina 27157
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
-2 region of human serum (6); a study of rabbit serum suggested that
2-macroglobulin has ferritin binding activity (7). The
role of ferritin-binding proteins(s) is uncertain; however, it has been
suggested that ferritin-binding proteins may serve as acquired
receptors for ferritin itself. This view is increasingly attractive in
light of recent reports documenting the presence of ferritin receptors
on the surface of cells (8-12) and the suggested role of this
interaction in numerous functions, including suppression of myelopoesis
(13), suppression of lymphocyte proliferation (14), and facilitation of
iron uptake (11). In order to begin to study the role of
ferritin-binding proteins and their potential relationship to ferritin
receptors, we have purified a ferritin-binding protein from human
serum. In this report, we describe the purification of this protein and
its identification as H-kininogen.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Detection of a ferritin-binding protein by
ligand blotting. A, 0.5 µl of human serum was applied to
seven lanes of a 7% SDS-PAGE gel, electrophoresed, and transferred to
nitrocellulose. The blot was cut into seven strips, each of which was
incubated separately. All lanes were blocked with nonfat milk.
Lane 1 was then incubated sequentially with spleen ferritin,
a monoclonal anti-ferritin antibody, HRP-conjugated anti-mouse
antibody, and a chemiluminescent substrate. Lanes 2-4 were
incubated in the same way, except that ferritin, anti-ferritin
antibody, and HRP-conjugated anti-mouse antibody, respectively, were
individually omitted. Lane 5 was incubated with ferritin,
HRP-conjugated anti-ferritin antibody, and a chemiluminescent
substrate. Lanes 6 and 7 were incubated in the
same way, except that ferritin and HRP-conjugated anti-ferritin
antibody, respectively, were individually omitted. Lanes
1-4 were exposed to film for 60 s; lanes 5-7
were exposed for 1 s. B, quantitation of ferritin
binding activity in serum. Human serum was diluted 1:10 in PBS, and 0, 1, 2, 4, 6, and 8 µl were analyzed by ligand blotting using
HRP-conjugated anti-ferritin antibody. Signal intensities were
quantitated by scanning densitometry. Ferritin-binding proteins appear
as multiple or single bands depending on the extent of spontaneous
proteolysis of the high molecular weight band, as discussed in the text
(see under "Discussion").
Purification of a Ferritin-binding Protein-- In order to purify ferritin binding activity, ammonium sulfate was added to 5 ml of freshly collected human serum to 50% saturation. The precipitate was dissolved in sodium acetate buffer (pH 5.8) containing 0.1 M sodium chloride and applied to a CM-cellulose column. As seen in Fig. 2A, the majority of the protein eluted in the void volume. Ferritin binding activity was retained in the column and was eluted by applying a 0.1-0.7 M sodium chloride gradient. Active fractions were pooled and concentrated, resulting in a 232-fold purification with 26% yield.
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Human H-kininogen Possesses Ferritin Binding Activity-- Despite these results, it remained possible that multiple proteins were present in the 100-kDa region of the PVDF membrane and that ferritin binding activity was attributable to the presence of a protein co-migrating with H-kininogen in this region of the gel. To verify that human H-kininogen possessed ferritin binding activity, commercially purified H-kininogen was subjected to SDS-PAGE and analyzed by ligand blotting. This protein was >95% pure and was purified using a procedure different from that described above. As shown in Fig. 2C, commercially purified H-kininogen showed ferritin binding activity on a ligand blot. Further, the mobility of the kininogen species detected by Western blotting using an anti-kininogen antibody was identical to those detected using the ferritin-binding protein ligand blot and corresponded to two of the three bands found in human serum (the higher molecular weight H-kininogen band seen in the serum sample was not present in the preparation of H-kininogen shown in Fig. 2C). Thus, purified H-kininogen also possesses ferritin binding activity.
Kininogen Binds to Recombinant H and L Ferritins-- Ferritin is a 24-subunit protein composed of two different types of subunits, termed H and L. The ratio of these subunits in ferritins isolated from different tissues varies widely, ranging from proteins that are rich in the H subunit (such as those found in the human heart) to those of predominantly L subunit. Although the identity of serum ferritin remains uncertain, it cross-reacts with antibodies directed against human spleen ferritin, and is therefore thought to contain L-type ferritin subunits, as well as an immunologically related glycosylated G subunit (25). In order to determine whether H-kininogen preferentially binds to a particular isoform of ferritin, we assessed its ability to bind to recombinant ferritins composed exclusively of either the H or L type subunit. As shown in Fig. 3A, both recombinant H and L ferritins were able to bind to H-kininogen. This suggests that H-kininogen binds to ferritin via a domain conserved between ferritin H and L.
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Deficiency of Ferritin Binding Activity in H-kininogen-deficient Plasma-- Kininogen deficiency is a rare genetic trait (26, 27). The existence of kininogen deficiency syndromes allowed us to test whether ferritin binding activity and H-kininogen were genetically as well as biochemically related: if ferritin binding activity is attributable to human H-kininogen, we would predict that it should not be detectable in plasma obtained from H-kininogen-deficient individuals. In order to test this hypothesis, levels of ferritin binding activity in pooled plasma from normal donors and kininogen-deficient individuals were compared. As shown in Fig. 3B, ferritin binding activity could not be detected in ligand blots of kininogen-deficient plasma, although it could readily be detected in pooled normal plasma. Western blotting with anti-kininogen antibodies confirmed that kininogen-deficient plasma contained greatly reduced levels of H-kininogen when compared with normal plasma (Fig. 3B).
H-kininogen and Ferritin Associate as Native Proteins-- We next wished to determine whether ferritin binding activity was a property of native as well as denatured kininogen. To address this issue, serum was applied to a nondenaturing gel, and a ligand blot assay was performed. As shown in Fig. 3C, ferritin binding activity was preserved in the absence of detergent treatment, suggesting that kininogen in its native conformation maintains ferritin binding activity.
The Light Chain of H-kininogen Contains the Ferritin Binding Domain-- H and L kininogens are related proteins derived by alternative splicing from a single gene (28). They share an identical N-terminal heavy chain but have different C-terminal light chains. In order to map the domain of H-kininogen responsible for ferritin binding, we tested whether ferritin binding was also exhibited by L-kininogen. As shown in Fig. 3D, when purified H- and L-kininogens were simultaneously analyzed for ferritin binding by ligand blotting, ferritin binding was only seen in lanes containing H-kininogen. Because the heavy chain of both H- and L-kininogen are identical, this result maps the domain important for ferritin interaction to the light chain of H-kininogen.
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DISCUSSION |
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We describe here the detection and isolation of a ferritin-binding protein from human serum. Several lines of evidence indicate that this protein is identical to human H-kininogen. First, partial amino acid sequencing of a protein that was isolated based on ferritin binding activity revealed identity to 16 amino acids present in domain 5 of human H-kininogen. Although our results do not exclude the possibility that the ferritin-binding protein may be a variant of H-kininogen with a small difference in primary amino acid sequence, we have no positive evidence to suggest that this is the case. This biochemical evidence was supported by the observation that plasma from individuals deficient in H-kininogen was also deficient in ferritin binding activity. A genetically determined deficiency in H-kininogen thus resulted in a concomitant reduction in ferritin binding activity.
The number and pattern of ferritin-binding proteins seen in these experiments was consistent with the known processing pathway for H-kininogen. Cleavage of H-kininogen by plasma kallikrein follows a reaction sequence in which the precursor is nicked twice to release the internal peptide bradykinin. This creates a two-chain H-kininogen intermediate in which the heavy and light chains remain linked by a disulfide bond. A subsequent cleavage then releases an additional 8-kDa peptide from the light chain. H-kininogen and its cleavage products thus appear as three distinct species on nonreducing gels, with the smallest species becoming most abundant as digestion with kallikrein proceeds (29-33). This pattern was also seen in our experiments, for both H-kininogen and ferritin-binding protein (e.g. Fig. 1A and Fig. 2C). The variation in number and intensity of these bands in different preparations likely reflects variability in the degree of spontaneous cleavage and processing that occurred during preparation of different batches of serum, which we did not attempt to control. Under our electrophoretic conditions, both ferritin-binding proteins and H-kininogen were seen to migrate with apparent molecular masses of approximately 140, 120, and 100 kDa, mobilities that exactly duplicate those reported by others for H-kininogen and its cleavage products (see, for example, Refs. 30-32) (although some groups have reported mobilities of H-kininogen cleavage products that more closely approximate the predicted molecular weights of these species (29, 33)). N-terminal sequence analysis was also consistent with the interpretation that the 100-kDa ferritin-binding protein represented an H-kininogen cleavage product because the sequence obtained mapped within one amino acid of the N terminus of the H-kininogen light chain.
The ligand blotting assay has been frequently used in the detection of receptor/ligand interactions (22-24). However, this assay measures the binding of a ligand to SDS-denatured proteins immobilized on nitrocellulose, a process that may have the potential to artifactually unmask binding sites not present in the native protein. We therefore confirmed that ferritin binding activity was retained in samples electrophoresed under nondenaturing conditions (Fig. 3C). Because native, as well as denatured, H-kininogen possessed ferritin binding activity, we conclude that ferritin and H-kininogen interact as native proteins.
H-kininogen is a large protein composed of one 362-amino acid heavy chain, the 9-residue bradykinin sequence, and one 255-amino acid light chain (28). The multiple functions of H-kininogen have been mapped to several domains within these chains (reviewed in Ref. 15). The N-terminal heavy chain, for example, contains three domains of cystatin-like structure, which mediate the cysteine protease inhibitor function of H-kininogen, whereas the light chain contains the surface binding site important in contact activation, as well as binding sites for prekallikrein and factor XI. Domains in the light chain also contribute to cell surface binding (18, 34, 35). To determine which region of the kininogen protein contains the ferritin binding domain, we tested whether L-kininogen, a protein with an identical heavy chain but different light chain, exhibited ferritin binding activity. The results (Fig. 3D) indicate that ferritin binding is contained within the light chain of H-kininogen and suggest that ferritin may particularly target light chain-dependent functions, such as binding to kallikrein or cell surfaces (35).
The interaction between ferritin and H-kininogen in human serum was
very specific, and using this assay, we could not detect binding to
other serum proteins. It is possible that other ferritin-binding proteins, which have been suggested to associate with ferritin (e.g. 2-macroglobulin (7)), are not detected
under our assay conditions, particularly if they associate with
ferritin as multimeric protein complexes (6). The possibility that
H-kininogen may participate in such higher order complexes is also not
excluded.
Previous experiments have shown that treatment of serum with agents that preserve ferritin structure but permit its dissociation from ferritin-associated proteins (e.g. heat, detergent) increase ferritin levels as measured by immunoassay (36). This suggests that binding of ferritin to ferritin-binding proteins occurs in vivo and may affect clinical assessment of serum ferritin levels.
One implication of the identification of kininogen as a ferritin-binding protein relates to numerous reports of a ferritin receptor (8-12) and to the possibility that a ferritin-binding protein might represent a shed form of a ferritin receptor. Although we have no evidence implicating kininogen as a cell-bound ferritin receptor, kininogen has been reported in many tissues (37), and our results are not incompatible with the hypothesis that kininogen docked on the surface of cells may function as an acquired ferritin receptor. Experiments to test this possibility are currently in progress.
Human ferritin L and H chains exhibit 56% amino acid identity (38) and
share a similar secondary structure comprised of five helical
domains and an intervening loop (1). Thus, despite their functional
differences (1, 3, 21), ferritin H and L subunits display some closely
similar domains, as emphasized by the isolation of monoclonal
anti-ferritin antibodies that recognize both the ferritin H and L
subunits (39). As shown in Fig. 3A, H-kininogen can bind to
both recombinant H and recombinant L ferritin, and therefore recognizes
a domain shared by these two proteins. Because H and L ferritins differ
in isoelectic point (H is more acidic than L), this result also
indicates that the overall charge of ferritin does not significantly
influence binding to H-kininogen.
What is the availability of ferritin for participation in complex formation in vivo? Based on its distribution in the body, there are two potential sources of ferritin: serum and tissue. In these experiments, we used ferritin purified from human spleen as well as recombinant tissue-type L and H ferritin to detect ferritin-binding proteins in serum (Fig. 3A). Thus, H-kininogen has the capacity to bind tissue ferritin and may do so at sites of localized release (for example, from macrophages (40)). Additionally, serum ferritin possesses immunological cross-reactivity with L type tissue ferritin (25) and may bind to H-kininogen as well. Others have suggested that ferritin-binding proteins may bind to serum ferritin more avidly than to exogenously added tissue ferritin (36). Nevertheless, except under unusual physiological circumstances, the concentration of serum ferritin is generally low relative to that of H-kininogen. Thus, at present, we favor a hypothesis that the interaction between H-kininogen and ferritin may take place at localized sites, for example, at cell surfaces, sites at which H-kininogen and ferritin have both been individually observed (8-12, 17-19). Such a mechanism might lead to a paracrine-like effect similar to that exhibited by certain cytokines (e.g. interleukin-1 and interleukin-6), hormones, and growth factors.
Although further experiments will be required to define the role of the ferritin/H-kininogen complex, both proteins have been linked to the inflammatory response. Thus, in addition to its role in contact activation and protease inhibition, H-kininogen is also the precursor of bradykinin, a potent proinflammatory peptide, the release of which is triggered by trauma, infection, and allergic reactions. Bradykinin in turn stimulates the release of further inflammatory mediators, including prostaglandins, tumor necrosis factor, and interleukins (41). Serum ferritin is up-regulated in acute and chronic inflammation, and both serum and tissue ferritins have been shown to be responsive to induction by inflammatory mediators (42-44). Thus, the interaction between ferritin and H-kininogen may represent a nodal point by which localized effects of H-kininogen, including release of bradykinin, may be modulated. Further experiments will be required to test these hypotheses.
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ACKNOWLEDGEMENTS |
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We thank Qixu Liu and Rong Ma for superb technical assistance. We are grateful to Karen Craver of the Special Hematology Laboratory of the Wake Forest University School of Medicine for kininogen-deficient plasma and normal reference plasma and to Drs. John Owen and Roy Hantgan for careful reading of the manuscript. Protein sequencing was performed in the Protein Analysis Core Laboratory of the Comprehensive Cancer Center of Wake Forest University.
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FOOTNOTES |
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* Protein sequencing was supported in part by National Institutes of Health Grants CA-12197 and RR-04869 and by a grant from the North Carolina Biotechnology Center. This work was supported in part by National Institutes of Health Grant DK 42412.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-9357; Fax: 336-716-7671.
1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride.
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REFERENCES |
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