An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m300456200
ISSN1083-351X
AutoresXinle Cui, Farshid N. Rouhani, Feras Hawari, Stewart J. Levine,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoAminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the type I tumor necrosis factor receptor (TNFR1) and promotes receptor shedding. Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and the interleukin-6 receptor (IL-6Rα), we hypothesized that ARTS-1 might also regulate shedding of IL-6Rα, a member of the type I cytokine receptor superfamily that is structurally different from TNFR1. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6Rα, a size consistent with soluble IL-6Rα generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6Rα shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6Rα shedding, and decreased membrane-associated IL-6Rα in cell lines overexpressing ARTS-1. The absence of basal IL-6Rα shedding from arts-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6Rα shedding. Furthermore, the mechanism of constitutive IL-6Rα shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6Rα) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multifunctional aminopeptidase that may modulate inflammatory events by promoting IL-6Rα and TNFR1 shedding. Aminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the type I tumor necrosis factor receptor (TNFR1) and promotes receptor shedding. Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and the interleukin-6 receptor (IL-6Rα), we hypothesized that ARTS-1 might also regulate shedding of IL-6Rα, a member of the type I cytokine receptor superfamily that is structurally different from TNFR1. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6Rα, a size consistent with soluble IL-6Rα generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6Rα shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6Rα shedding, and decreased membrane-associated IL-6Rα in cell lines overexpressing ARTS-1. The absence of basal IL-6Rα shedding from arts-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6Rα shedding. Furthermore, the mechanism of constitutive IL-6Rα shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6Rα) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multifunctional aminopeptidase that may modulate inflammatory events by promoting IL-6Rα and TNFR1 shedding. Although interleukin-6 (IL-6) 1The abbreviations used are: IL-6, interleukin-6; IL-6Rα, interleukin-6 α-receptor; ARTS-1, aminopeptidase regulator of TNFR1 shedding; TNFR1, type I tumor necrosis factor receptor; sIL-6Rα, soluble IL-6 receptors; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; STAT, signal transducer and activator of transcription; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high pressure liquid chromatography; RT, reverse transcriptase; gp, glycoprotein; sgp130, soluble form of gp130; TACE, TNF-α-converting enzyme; PMA, phorbol 12-myristate 13-acetate.1The abbreviations used are: IL-6, interleukin-6; IL-6Rα, interleukin-6 α-receptor; ARTS-1, aminopeptidase regulator of TNFR1 shedding; TNFR1, type I tumor necrosis factor receptor; sIL-6Rα, soluble IL-6 receptors; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; STAT, signal transducer and activator of transcription; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high pressure liquid chromatography; RT, reverse transcriptase; gp, glycoprotein; sgp130, soluble form of gp130; TACE, TNF-α-converting enzyme; PMA, phorbol 12-myristate 13-acetate. was originally identified as a B cell differentiation factor, it is now recognized to function as a pleiotropic cytokine capable of modulating a variety of immune and inflammatory responses (e.g. the hepatic acute phase response, T cell activation, bone metabolism, and hematopoiesis) (1Jones S.A. 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Novick D. Yamamoto N. Fuller G.M. Eur. J. Immunol. 1998; 28: 3514-3522Crossref PubMed Scopus (59) Google Scholar, 26Gallea-Robache S. Morand V. Millet S. Bruneau J.M. Bhatnagar N. Chouaib S. Roman-Roman S. Cytokine. 1997; 9: 340-346Crossref PubMed Scopus (72) Google Scholar, 27Mullberg J. Durie F.H. Otten-Evans C. Alderson M.R. Rose-John S. Cosman D. Black R.A. Mohler K.M. J. Immunol. 1995; 155: 5198-5205PubMed Google Scholar, 28Hargreaves P.G. Wang F. Antcliff J. Murphy G. Lawry J. Russel R.G.G. Croucher P.I. Br. J. Haematol. 1998; 101: 694-702Crossref PubMed Scopus (70) Google Scholar). Further evidence supporting TACE-mediated IL-6Rα shedding is the strong reduction in phorbol ester-induced IL-6Rα shedding in TACE-deficient murine fibroblasts that can be rescued by reconstitution of TACE expression (25Althoff K. Reddy P. Voltz N. Rose-John S. Mullberg J. Eur. J. Biochem. 2000; 267: 2624-2631Crossref PubMed Scopus (146) Google Scholar). However, the existence of additional IL-6Rα sheddases has been suggested by a basal hydroxamate-sensitive IL-6Rα shedding from TACE-deficient cells, as well as by IL-6Rα shedding that is resistant to the hydroxamic acid-based metalloprotease inhibitor, TAPI (9Jones S.A. Novick D. Horiuchi S. Yamamoto N. Szalai A.J. Fuller G.M. J. Exp. Med. 1999; 189: 599-604Crossref PubMed Scopus (148) Google Scholar, 14Vermes C. Jacobs J.J. Zhang J. Firneisz G. Roebuck K.A. Glant T.T. J. Biol. Chem. 2002; 277: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Althoff K. Reddy P. Voltz N. Rose-John S. Mullberg J. Eur. J. Biochem. 2000; 267: 2624-2631Crossref PubMed Scopus (146) Google Scholar). The aminopeptidase regulator of TNFR1 shedding (ARTS-1) has recently been identified as a type II integral membrane protein that binds to the TNFR1 extracellular domain and promotes TNFR1 shedding (29Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (189) Google Scholar). Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and IL-6Rα, we hypothesized that ARTS-1 might also regulate IL-6Rα shedding. By utilizing an arts-1 knock-out cell line (arts-1 (–/–)), we demonstrate that ARTS-1 is required for constitutive IL-6Rα shedding. Transfection of arts-1 (–/–) cell lines with plasmids containing full-length ARTS-1 restored IL-6Rα shedding, whereas transfection with ARTS-1 catalytic site mutants did not. These data indicate that the mechanism of constitutive IL-6Rα shedding requires ARTS-1 catalytic activity. Furthermore, ARTS-1 directly binds to a 55-kDa IL-6Rα, a size consistent with soluble IL-6Rα generated by ectodomain cleavage of the membrane-bound receptor. Thus, ARTS-1 may modulate inflammatory events by promoting the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6Rα) and the TNF receptor superfamily (TNFR1). ARTS-1/IL-6Rα Co-immunoprecipitation and Immunoblotting—The NCI-H292 human pulmonary mucoepidermoid carcinoma cell line was purchased from the American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% fetal bovine serum under 5% CO2 at 37 °C. Immunoprecipitation experiments were performed as described previously (29Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (189) Google Scholar). Briefly, cells were harvested for membrane isolation by scraping and were disrupted by sonicating twice (for 10 s each) in lysis buffer (50 mm Tris-HCl, pH 7.2, 120 mm NaCl, 0.1% Triton X-100, and Complete™ protease inhibitor (Roche Applied Science)), followed by centrifugation at 1,000 × g for 5 min to remove nuclei and cellular debris. Post-nuclear supernatants were centrifuged at 100,000 × g for1hto recover membrane pellets that were suspended by sonicating three times (for 2 s each) in lysis buffer. Protein concentrations were determined utilizing the BCA protein determination kit (Pierce). For immunoprecipitation, samples of membrane proteins (200 μg) were incubated overnight, 4 °C, with 20 μg of murine monoclonal anti-human IL-6Rα antibody against the IL-6Rα extracellular domain (R & D Systems) or 1 μl of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 μl of immobilized protein A/G beads (Pierce) for 2 h at room temperature. The ARTS-1 antibody recognizes a 17-amino acid epitope (RGRNVHMKQE-HYMKGSD) located in exon 11 (29Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (189) Google Scholar). After the beads were washed 8 times, bound proteins were separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated overnight (4 °C) with ARTS-1 immune or pre-immune serum diluted 1:20,000 or the murine anti-IL-6Rα monoclonal antibody, 2 μg/ml. Detection was by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies. For immunoblotting, samples of membrane proteins (20–40 μg) were separated via SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated overnight (4 °C) with either ARTS-1 immune serum, diluted 1:20,000, rabbit polyclonal anti-human IL-6Rα (Santa Cruz Biotechnology), 1 μg/ml, or goat anti-MUC1 polyclonal antibody (Santa Cruz Biotechnology), 1 μg/ml. Detection was by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies. ARTS-1 Cell Lines—Stably transfected NCI-H292 cell lines that expressed either full-length human ARTS-1 or antisense ARTS-1 (bases 61–213) were utilized, as described previously (29Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (189) Google Scholar). To quantify membrane-associated IL-6Rα, membrane fractions were prepared, as described above, and assays were performed on 300 μg of membrane proteins utilizing a sandwich ELISA with a sensitivity of 7.8 pg/ml (R & D Systems). sIL-6Rα in cell culture supernatants was assayed by ELISA. Immunoblotting was performed on culture supernatants that were concentrated 30-fold utilizing a Centriprep filter with a 10-kDa exclusion (Amicon). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. TAPI-0, TAPI-1, and TAPI-2 were purchased from Peptides International. Statistical analysis was performed using a Student's t test with a Bonferroni correction for multiple comparisons. A p value of less than 0.05 was considered significant. Construction of an arts-1 Knock-out NCI-H292 Cell Line—The arts-1 targeting vector was generated by assembling sequences flanking arts-1 exons 5 and 6 as the upstream and downstream arms of the PKO scrambler NTKV 1902 vector (Stratagene) (30Hattori A. Matsumoto K. Mizutani S. Tsujimoto M. J. Biochem. (Tokyo). 2001; 130: 235-241Crossref PubMed Scopus (44) Google Scholar). The following PCR primer pairs, which span exons 3 and 4, were utilized to generate the up-stream arm: primer A (5′-CCC-AAG-CTT-GGG-TTC-TCC-CTC-TGT-TAG-TCG-C-3′) and primer B (5′-CCA-TCG-ATG-GTG-AAA-TGA-CAG-TTA-GAC-CCT-C-3′). Primer A contains a HindIII restriction site, whereas primer B contains a ClaI restriction site. The downstream arm, which spans exons 7 and 8, was generated utilizing the following primers: primer C (5′-CGG-GAT-CCC-GAT-TGT-TTC-TCC-AAA-GCA-TTC-GT-3′), which contains a BamHI restriction site, and primer D (5′-TCC-CCC-GGG-GGA-CAT-CAT-CTG-CCA-ACT-CCC-TTT-G-3′), which contains a SmaI restriction site. Genomic DNA isolated from NCI-H292 cells was utilized as a template for PCR amplification of the 7468-bp upstream segments and the 2783-bp downstream segments utilizing Pfu turbo DNA polymerase (Stratagene). The PCR products were digested with the appropriate endonucleases to generate the 3841-bp upstream arm, based upon the presence of an internal HindIII restriction site, and the 2783 downstream arm. The cDNA segments were gel-purified and ligated into the polylinker region of the PKO scrambler NTKV 1902 vector, which contains both positive (neomycin phosphotransferase) and negative (thymidine kinase) selection markers. The neomycin phosphotransferase gene, in the antisense orientation, is driven by a phosphoglycerate kinase promoter, whereas the thymidine kinase gene, in the sense orientation, is driven by a polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. Sequences of the upstream and downstream arms were confirmed by DNA sequencing. NCI-H292 cell lines were transfected with the arts-1 targeting vector using FuGENE 6 (Roche Applied Science) and maintained under selective pressure by addition of 20 mg/ml geneticin (Invitrogen) and 400 μm ganciclovir (Sigma) to medium followed by pH adjustment with NaHCO3. Transfected cells were cloned by limiting dilution, and clones containing homozygous deletions generated by homologous recombination were characterized by PCR and RT-PCR analysis of genomic DNA and mRNA utilizing the following primer pairs: Neo 1 sense, 5′-TTC-CTT-CCC-TGG-CAT-CTA-CCT-C-3′, and arts-1 intron 8/9 antisense, 5′-TTC-CCG-CTT-CAG-TGA-CAA-CG-3′; Neo 2 sense, 5′-TCG-CCT-TCT-ATC-GCC-TTC-TTG-3′, and arts-1 intron 10/11 antisense, 5′-AAA-AGA-ATG-TGC-TTG-GGG-GAA-C-3′; arts-1 exon 5/6 sense, 5′-CAG-TCA-TTG-TGA-TGC-CAA-G-3′, and arts-1 exon 5/6 antisense, 5′-GCT-GTG-CCA-GAC-AAG-ATA-AAT-C-3′; arts-1 exon 15/17 sense, 5′-CTA-CTG-GGT-TCC-TGC-CAA-TGA-G-3′, and arts-1 exon 15/17 antisense, 5′-TCA-ACT-ACT-ACT-CCT-CGC-CTG-TGT-G-3′; arts-1 exon 2/19 sense, 5′-CAT-GGT-GTC-AGA-GCA-CT-3′, and arts-1 exon 2/19 antisense, 5′-CAT-ACG-TTC-AAG-CTT-TTC-3′; G3PDH sense, 5′-TGA-AGG-TCG-GAG-TCA-ACG-GAT-TTG-GT-3′, and G3PDH antisense, 5′-CAT-GTG-GGC-CAT-GAG-GTC-CAC-CAC-3′. RT-PCR of IL-6 mRNA was performed utilizing previously described primer pairs that span the IL-6Rα transmembrane domain to generate a 398-bp product (24Horiuchi S. Koyanagi Y. Zhou Y. Miyamoto H. Tanaka Y. Waki M. Matsumoto A. Yamamoto M. Yamamoto N. Eur. J. Immunol. 1994; 24: 1945-1948Crossref PubMed Scopus (185) Google Scholar). The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers were purchased from Clontech. arts-1 (–/–) cell lines were reconstituted by transient transfection with plasmids encoding either full-length ARTS-1 or ARTS-1 catalytic site mutants (H353P, H357V, and H353P/E354V), utilizing Gene Porter II and Booster (Gene Therapy Systems), as described previously (29Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (189) Google Scholar). IL-6Rα Ectodomain Cleavage Assay—A model system was utilized to assess whether ARTS-1 catalyzes the proteolytic cleavage of the IL-6Rα ectodomain. A recombinant glutathione S-transferase-ARTS-1 (GST-ARTS-1) fusion protein was synthesized in BL21 Escherichia coli transfected with a pGEX-6P-1 plasmid encoding the ARTS-1 extracellular domain and purified using glutathione-Sepharose 4B (Amersham Biosciences) (29Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (189) Google Scholar). The GST-ARTS-1 was recovered from the insoluble fraction by denaturation with 6 m urea in phosphate-buffered saline and refolded by serial dialysis against phosphate-buffered saline containing decreasing urea concentrations. The GST-ARTS-1 fusion protein was demonstrated to be catalytically active and possess aminopeptidase activity against leucine, methionine, alanine, and phenylalanine p-nitroanilide model substrates. A 22-amino acid model peptide substrate (RDSANATSLPVQDSSSVPLPTF) containing the IL-6Rα cleavage site was synthesized by Sigma-Genosys. Peptides corresponding to the 12-amino acid N-terminal cleavage product (RDSANATSLPVQ) and the 10-amino acid C-terminal cleavage product (DSSSVPLPTF) were also synthesized to serve as standards. The peptide substrate (2.5 μg/ml) was incubated with GST-ARTS-1 (200 ng/ml) for 2 h at 37 °C before transfer of 50 μl of reaction mixture to a Vydac (Hesperia, CA) C18 column (The Nest Group, Southborough, MA) equilibrated with solution A (0.1% trifluoroacetic acid, HPLC grade water). The mixture was then separated by gradient elution with solution B (0.1% trifluoroacetic acid, acetonitrile) at a flow rate of 0.8 ml/min: 100% solution A, 0–2 min, linear gradient 0–67.5% of solution B, 2–20 min. The absorbance of the eluate was recorded at 214 nm. Membrane-associated ARTS-1 Binds to IL-6Rα—Immunoprecipitation experiments were performed to assess whether an endogenous protein-protein interaction exists between ARTS-1 and IL-6Rα in NCI-H292 cells. As shown in Fig. 1A, immunoprecipitation of membrane fractions with an anti-IL-6Rα monoclonal antibody pulled down the 100-kDa ARTS-1 protein. In the reciprocal experiment (Fig. 1B), immunoprecipitation with ARTS-1 antiserum pulled down a 55-kDa IL-6Rα species, which is consistent with the soluble form of IL-6Rα generated by proteolytic cleavage of the IL-6Rα extracellular domain. These experiments demonstrate that the 100-kDa membrane-associated ARTS-1 species binds the 55-kDa soluble form of IL-6Rα. Because both IL-6Rα and TNFR1 can be co-immunoprecipitated with ARTS-1, we next assessed whether the binding of IL-6Rα and TNFR1 to ARTS-1 is mutually exclusive. When immunoblots demonstrating that an anti-IL-6Rα antibody coimmunoprecipitates ARTS-1 were stripped and re-probed with an anti-TNFR1 antibody, no TNFR1 was detected. Similarly, immunoblots demonstrating that an anti-TNFR1 antibody coimmunoprecipitates ARTS-1 were stripped and re-probed with an anti-IL-6Rα antibody, no IL-6Rα was detected (data not shown). When immunoblots demonstrating that an anti-ARTS-1 antibody co-immunoprecipitates IL-6Rα were stripped and reprobed with an anti-TNFR1 antibody, TNFR1 was detected. These experiments are consistent with the conclusion that the bindings of IL-6Rα and TNFR1 to ARTS-1 are mutually exclusive. ARTS-1 Promotes IL-6Rα Shedding—To determine whether ARTS-1 increases IL-6Rα shedding, experiments were performed utilizing cell lines stably transfected with ARTS-1 cDNA in either the sense or antisense orientation (29Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (189) Google Scholar). The ARTS-1 cell lines express full-length ARTS-1 coding sequence, whereas the antisense cell lines express ARTS-1 bases 61–213, which includes the putative translation start site and the intracellular and transmembrane domains. The effect of ARTS-1 protein expression on IL-6Rα ectodomain shedding into culture supernatants was assessed by ELISA. As shown in Fig. 2A, the amount of sIL-6Rα present in culture supernatants from cell lines overexpressing ARTS-1 was significantly greater than that of mock-transfected cells, whereas supernatants from cell lines transfected with antisense ARTS-1 had significantly less sIL-6Rα than did mock-transfected cells. The quantity of soluble 55-kDa IL-6Rα in culture supernatants was also analyzed by immunoblotting. As shown in Fig. 2B, the quantity of 55-kDa IL-6Rα in culture supernatants from ARTS-1 cell lines was significantly greater than that from wild type NCI-H292 cells or mock-transfected cells. Reciprocally, the quantity of IL-6Rα protein in supernatants from ARTS-1 antisense cell lines was markedly less. These experiments demonstrate that changes in ARTS-1 protein levels correlated directly with changes in soluble IL-6Rα protein. Experiments were also performed to assess whether ARTS-1 protein expression correlated with levels of membrane-associated IL-6Rα. As shown in Fig. 2C, the quantity of membrane-associated IL-6Rα, as determined by ELISA, was significantly greater in the ARTS-1 antisense cell lines than in mock-transfected cell lines, whereas the quantity of membrane-associated IL-6Rα in the ARTS-1 cell lines was below the limit of detection. Similarly, as shown in Fig. 2D, the quantity of full-length 80-kDa IL-6Rα, as determined by immunoblotting, was significantly greater in the ARTS-1 antisense cell lines than in mock-transfected cell lines, whereas the quantity of membrane-associated full-length 80-kDa IL-6Rα in the ARTS-1 cell lines was significantly reduced. These experiments demonstrate that ARTS-1 protein expression is inversely correlated with levels of membrane-associated IL-6Rα, consistent with the ability of ARTS-1 to promote IL-6Rα shedding. Further, in contrast to the ARTS-1 pull-down experiments, no 55-kDa, cleaved IL-6Rα was identified in crude membrane fractions. This suggests that IL-6Rα ectodomain cleavage and binding to ARTS-1 require co-localization within cellular membranes, as occurs during immunoprecipitation. NCI-H292 Cells Selectively Express the Membrane-Bound IL-6 Receptor—RT-PCR experiments were performed to characterize the expression of IL-6Rα isoforms by NCI-H292 cells. Primers were utilized that spanned the IL-6Rα transmembrane domain and could amplify mRNAs for both the membrane-bound IL-6Rα species, as demonstrated by a 398-bp PCR product, and the alternatively spliced, soluble IL-6Rα species, as demonstrated by a 304-bp product (24Horiuchi S. Koyanagi Y. Zhou Y. Miyamoto H. Tanaka Y. Waki M. Matsumoto A. Yamamoto M. Yamamoto N. Eur. J. Immunol. 1994; 24: 1945-1948Crossref PubMed Scopus (185) Google Scholar). As shown in bottom right panel of Fig. 3B, RT-PCR amplification of NCI-H292 cell mRNA revealed a single 398-bp PCR product that is consistent with expression of the membrane-bound IL-6Rα. I
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