Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Affinity kinetics between hIL-6 mutants and hIL-6R − gp130 assayed by SPR. gp130 was immobilized onto a CM5 chips via standard amine coupling and different concentrations of hIL-6 mutants with a saturated concentration of hIL-6R were pre incubated and the mixture were injected. Affinity kinetics were analyzed by the Langmuir’s 1:1 model fit on seven serial dilutions of hIL-6 mutants from 250 nM and flow speed of 30 μL/min. ( A ) Schematic diagram of binding affinity between hIL-6 mutants and hIL-6R − gp130 by SPR; ( B – K ) representative examples of curve fits for the affinity kinetic analysis of hIL-6 mutants.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques: Concentration Assay, Incubation, Injection, Binding Assay

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Affinity of the interaction between hIL-6/mutants and hIL-6R/gp130 by Biacore SPR.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques: Mutagenesis, Binding Assay

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: The spatial arrangement of amino acid residues hIL-6 R167 and E171 in the hexameric hIL-6 − hIL-6R − gp130 complex (PDB code: 1p9m). ( A ) Close spatial proximity is observed between hIL-6 R167 and E171 and the hIL-6 AB-loop; ( B ) the potential influence of hIL-6 R167 on the interaction between hIL-6 K53 and hIL-6R Q95 is indicated. The hIL-6 molecule is shown in brown, hIL-6R in purple, and gp130 in green.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques:

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 2. The expression of selected receptors (CXCR4, Leptin R, EGFR, IL2R gamma, and gp130) on cell membrane of selected breast cancer cell lines. gp130 was the only receptor that is overexpressed in selected triple negative breast cancer (TNBC) cell lines, MDA 231wild type (WT), MDA 231 K, and MDA 468 cell lines. The expression of the receptors was detected using fluorescent-labeled monoclonal antibodies to the selected receptors and flow cytometry. Asterisks indicates significant difference to non-TNBC cell lines (one-way ANOVA, Tukey post-hoc test; p<0.05). Bar graph represents the mean values (n = 4), and the error bars show the standard deviation.

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Expressing, Membrane, Labeling, Bioprocessing, Flow Cytometry, Standard Deviation

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 4. The effect of Individual and combinatorial silencing of selected proteins in JAK/STAT pathway on expression of selected antiapoptotic proteins (Bcl-2, Bcl-XL, and Mcl-1). The mRNA expression was determined using RT-PCR in MDA 231 (A) and MDA 468 cells (B). In both cell lines, the drop in mRNA level of all three antiapoptotic proteins was significantly higher in cells exposed to JAK2/gp130 siRNA combination. Bar graphs represent the mean values (n = 3), and the error bars show the standard deviation. The effect of single and combinational silencing of gp130 and JAK2 on Bcl2 and Mcl-1 at protein level was confirmed by western blotting (C) in MDA 231 cells. β-actin served as endogenous protein. A similar trend was noted at the protein level (D). Expression of antiapoptotic related molecules was normalized to untreated cells (NT). Bar graph represents the mean values (n = 3), and the error bars show the standard deviation. Asterisks indicates significant difference compared to all other study groups (one-way ANOVA, Tukey post-hoc test; p < 0.05).

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Western Blot

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 5. The effect of Individual and combinatorial silencing of selected proteins in JAK/STAT pathway on expression of selected pro-apoptotic proteins (PTEN and p53). The mRNA expression of the pro-apoptotic proteins was determined using RT-PCR in MDA-MB-231 (A) and MDA-MB-468 cells (B). The mRNA levels of both PTEN and P53 proteins showed the most significant increase in both cell lines after simultaneous silencing of JAK2/gp 130. Bar graphs represent the mean values (n = 3), and the error bars show the standard deviation. Th effect of single and silencing of gp130 and JAK2 on PTEN and P53 at protein level was confirmed by western blotting in MDA 231 cells (C). β-actin served as endogenous protein. A similar trend was noted at the protein level (D). Expression of pro-apoptosis proteins was normalized to untreated cells (NT). Bar graph represents the mean values (n = 3), and the error bars show the standard deviation. Asterisks indicates significant difference compared to all other study groups (one-way ANOVA, Tukey post-hoc test; p < 0.05).

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Western Blot

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 9. The effect of silencing STAT3 and the individual and combinational silencing of JAK2 and gp 130 on STAT3 activation in MDA 231 cell line using differential Western Blotting on cytoplasmic and nuclear compartments. GAPDH and Histone H3 levels were analyzed as cytoplasmic and nuclear markers, respectively, which indicated effective isolation of the sub-cellular compartments. The bands representing STAT3 show total STAT3 levels (phosphorylated and non-phosphorylated). (A) Western blot showing the cytoplasmic expression level of STAT3 and p-STAT3. The expression of p-STAT3 was negligible in cytoplasm compared to the total STAT3 levels. (B) Bar graph represents the cytoplasmic expression level of STAT3 in different study groups (n = 3; error bars represent standard deviation). The STAT3 levels in cytoplasm dropped most significantly as a response to silencing STAT3. (C) Differential western blot shows the nuclear expression level of STAT3 and p-STAT3. The effect of silencing is apparent on both total STAT3 and the p-STAT3 levels. (D) Bar graph shows nuclear protein levels of p- STAT3 in different study groups (n = 3; error bars represent standard deviation). The drop in level of p-STAT in nucleus was most significant in cells exposed to JAK2/gp130 siRNA combination.

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Activation Assay, Western Blot, Isolation, Expressing, Standard Deviation

Journal:

Article Title: Cloning and Characterization of a Specific Receptor for Mouse Oncostatin M

doi:

Figure Lengend Snippet: mOSM binds directly to either c12 or gp130. (A) Characterization of recombinant receptors produced in COS7 cells. COS7 cells were either mock transfected or transfected with gp130, c12, or a combination of c12 and gp130. Recombinant proteins were detected by Western blot analysis with antisera specific for either c12 (left) or gp130 (right). Molecular mass standards (in kilodaltons) are shown on the left and right. (B) COS cells transfected with either c12 or gp130 bind 125I-rmOSM. Parallel plates of transfected COS cells, as described in panel A, were tested for their ability to bind 125I-rmOSM. Shown are specifically bound cpm of 125I-rmOSM when tested at either 0.5 or 5 nM. Nonspecific binding represented 10 to 27% of total binding, depending on which receptor subunit was transfected and the concentration of the radiolabeled ligand. (C) 125I-rmOSM can be directly cross-linked to either c12 or gp130. Parallel plates of transfected COS cells, as described in panel A, were incubated with 125I-rmOSM in the presence (+) or absence (−) of a 100-fold molar excess of unlabeled rmOSM. The bound ligand was cross-linked to the receptors with DSS, the proteins were separated by SDS-PAGE, and the labeled complexes were visualized with a PhosphorImager. Molecular mass standards (in kilodaltons) are shown on the left.

Article Snippet: Anti-gp130 (M20), anti-STAT5B (C17), anti-STAT3 (C20), and anti-LIFR (C19) were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Recombinant, Produced, Transfection, Western Blot, Binding Assay, Concentration Assay, Incubation, SDS Page, Labeling

Journal:

Article Title: Cloning and Characterization of a Specific Receptor for Mouse Oncostatin M

doi:

Figure Lengend Snippet: hOSM and mOSM use different receptors in mouse cells. (A) NIH 3T3 cells were treated with cytokines to allow the detection of receptors present with anti-P.Tyr antibody. Immunoprecipitation (IP) of rmOSM-treated cells with antibody to c12 or of rmLIF-treated cells with antibodies to LIFRβ or gp130 revealed that all three were present as p180, p190, and p160 proteins, respectively. (B) NIH 3T3 cells were treated, lysed, and immunoprecipitated with anti-P.Tyr antibodies. The immunoprecipitates were analyzed by immunoblotting for the presence of STAT3, LIFR, or c12. (C) Phosphorylation and coimmunoprecipitation of all three receptor subunits were tested by treating NIH 3T3 cells with the indicated cytokine and immunoprecipitating each separately with antibodies to the indicated receptor subunit, followed by immunoblotting with anti-P.Tyr antibodies.

Article Snippet: Anti-gp130 (M20), anti-STAT5B (C17), anti-STAT3 (C20), and anti-LIFR (C19) were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: Cloning and Characterization of a Specific Receptor for Mouse Oncostatin M

doi:

Figure Lengend Snippet: Activation of receptor subunits and signaling molecules by OSM and LIF in primate and mouse cells

Article Snippet: Anti-gp130 (M20), anti-STAT5B (C17), anti-STAT3 (C20), and anti-LIFR (C19) were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 2. The expression of cell surface IL-6R and gp130 in MG-63 cultures af- ter different treatments. Semiconfluent osteoblast cultures were left untreated or treated with PMA or TNF- for 4 h, and the expression of cell surface IL-6R and gp130 were analyzed by flow cytometry. Closed his- tograms represent isotype antibody controls, and open histograms show either anti-IL-6R or anti-gp130 antibody-stained cells as indi- cated. The expression of IL-6R was reduced by both PMA and TNF-, whereas the level of gp130 was unaffected. The PMA-induced down-regulation of IL-6R expression was es- sentially abolished by staurosporine (stauro) and partially reversed by Galardin (Gal), whereas the suppressive effect of TNF- on IL-6R expression was substantially dimin- ished by UO126, a potent inhibitor of MEK1/MEK2.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Expressing, Flow Cytometry, Staining

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 5. The expression of cell sur- face IL-6R and gp130 in MG-63 cul- tures. Semiconfluent osteoblast cultures were left untreated or treated with differ- ent compounds for 4 or 24 h as indicated, and the expression levels of cell surface IL-6R and gp130 were analyzed by flow cytometry. Open histograms represent untreated cultures labeled with either an- ti-IL-6R or anti-gp130 antibody on each panel. Closed histograms show either iso- type antibody controls (panels labeled blank) or treated cultures (all other pan- els) labeled with either anti-IL-6R or anti- gp130 antibody as indicated in each panel. Note that the inhibitors of protein synthesis (cycloheximide (cyclohex)) and intracellular protein transport (brefeldin A (bref-A)) essentially diminished the ex- pression of both the IL-6R and gp130 on the cell surface after 4 h. Inhibition of transcription (actinomycin D (actino)) re- quired longer time periods to reduce the expression of either IL-6R or gp130.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Expressing, Flow Cytometry, Labeling, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 6. Western blot analysis of IL-6R and gp130 in MG-63 cells. Semiconfluent cell cultures were left untreated or treated with various compounds for different time periods as indicated, and the amounts of IL-6R (panels A and B, upper blots) and gp130 (panels A and B, lower blots) were analyzed. Cells were lysed, 700 g of protein of each cell lysate was incubated with either anti-IL-6R or anti-gp130 antibodies, and then immunocomplexes were precipitated and analyzed by Western blot. Membranes were immunoblotted with either anti-IL-6R or anti- gp130 antibodies. Transcription and translation inhibitors reduced the amounts of both IL-6R and gp130, whereas brefeldin A induced the accumulation of the low molecular mass forms of IL-6R (80 kDa) or gp130 (130 kDa) shown as var-IL-6R and var-gp130. Note that inhibi- tion of N-glycosylation by tunicamycin or treatment of samples with N-glycosidase F resulted in the 105-kDa band corresponding to gp130 (deglycos-gp130).

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Western Blot, Incubation, Glycoproteomics

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 7. Western blot analysis of tyrosine phosphorylation of gp130 in MG-63 cells (panel A) and bone marrow-derived pri- mary human osteoblasts (panel B). Semiconfluent cell cultures were left untreated or treated with various compounds as indicated. Cells were lysed, 700 g of protein was incubated with anti-gp130 antibody, and then immunocomplexes were precipitated and analyzed by Western blot. Membranes first were immunoblotted with anti-phosphotyrosine antibody (4G10, upper blot on each panel) and then stripped and re- blotted with anti-gp130 antibody (lower blot on each panel). Note that IL-6 and sIL-6R added together or conditioned medium (CM) of PMA- treated cultures induced tyrosine phosphorylation of gp130 (p-gp130). This effect was abolished by neutralizing antibodies to IL-6R. Addition- ally, note that two forms of gp130 can be detected (gp130 and var- gp130) and only the 145-kDa gp130 form was tyrosine-phosphorylated.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Western Blot, Phospho-proteomics, Derivative Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 8. Upper panel, analysis of IL-6, IL-6R, and gp130 mRNA expression by RPA is shown. Semiconfluent cultures of osteoblasts were left untreated or were treated with various compounds for different time periods as indicated. RNA was isolated from each sample, and RPA was performed. Constitutive mRNA expressions were observed for both IL-6R and gp130. mRNAs encoding IL-6, LIF, and GM-CSF were increased in TNF-- or LPS-treated cultures after 24 h. Lower panels, reverse transcription-PCR analysis of the IL-6R. Nucleotide sequence on panel C represents a segment (1381–1860 bp) of IL-6R (gp80, GenBank accession nos. X12830 and M20566) including the transmembrane encoding region (uppercase letters) and the 94-base pair deletion (exon 2, underlined part, missing in the 125-bp-long PCR product on panel B) of the alternative spliced variant (shown as DS-sIL-6R) (5). Boldface lowercase letters represent either the sense (1402–1421) or complementary antisense (1600–1620) sequences of primers used for PCR reactions. The boldface and underlined codons show the termination sites for the cognate (TAG) and the spliced variant (TGA) IL-6R mRNA.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Expressing, Isolation, Reverse Transcription, Sequencing, Variant Assay

Journal: Journal of neuropathology and experimental neurology

Article Title: Effects of oncostatin M on human cerebral endothelial cells and expression in inflammatory brain lesions.

doi: 10.1093/jnen/60.11.1087

Figure Lengend Snippet: Fig. 2. HCEC transcribe mRNA for gp130, OSMR-b, LIFR, and IL-6R, but not for CNTFR. RT-PCR was performed on RNA extracted from HCEC and reverse-transcribed (RT1) using oligo(dT) primers. RNA samples not submitted to reverse transcription were used as negative controls (RT2). PCR results for gp130, LIFR, and OSMR-b are shown in the upper panel. Note that the expression level of LIFR was below that of gp130 and OSMR-b even when a 3-fold (33) higher amount of cDNA was used as starting material in the RT-PCR. PCR amplicons of CNTFR, IL-6R and b-actin (b-A), which was used as an internal control, are shown in the lower panel. RNA extracted from early postmortem human brain (hu Brain) served as a positive control for CNTFR. Identical results were obtained in all 5 HCEC preparations analyzed. Length of bands from DNA size marker (M, 1 kb DNA ladder, Gibco BRL) is indicated on the left (A). Surface detection of gp130 and OSMR-b, but not of LIFR, on HCEC. FACS analysis was performed as described in Materials and Methods to determine surface expression of gp130, OSMR-b, and LIFR on HCEC. Histograms show stain- ing with the respective receptor antibody (shaded) underlaid with the isotype-matched control (single line). Hela cells were

Article Snippet: Human recombinant IL-6, LIF, OSM, and anti-human gp130 and LIFR neutralizing antibodies were obtained from R & D Systems (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Expressing, Control, Positive Control, Marker, Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: Effects of oncostatin M on human cerebral endothelial cells and expression in inflammatory brain lesions.

doi: 10.1093/jnen/60.11.1087

Figure Lengend Snippet: Fig. 5. OSM-induced upregulation of IL-6 is mediated via gp130. HCEC were preincubated with increasing concentrations (0.5–50 mg/ml) of neutralizing antibodies against gp130 or an isotype-matched control antibody (mouse IgG1k, 50 mg/ml) for 2 h at 378C. Subsequently, OSM (100 ng/ml) was added to cells for 20 h. Protein concentrations of IL-6 in supernatants were analyzed by ELISA. Results are expressed as the percentage of IL-6 production compared to the isotype control. Data are the mean 6 SEM from 3 experiments. *p , 0.05, ***p , 0.001.

Article Snippet: Human recombinant IL-6, LIF, OSM, and anti-human gp130 and LIFR neutralizing antibodies were obtained from R & D Systems (Minneapolis, MN).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta

Article Title: Trypanosoma cruzi, the causative agent of Chagas disease, modulates interleukin-6-induced STAT3 phosphorylation via gp130 cleavage in different host cells.

doi: 10.1016/j.bbadis.2012.12.003

Figure Lengend Snippet: Fig. 7. Cruzipain activity diminishes the gp130 D1 domain expression on human peripheral blood monocytes. Mononuclear cells were incubated with active cruzipain for 120 min. Then, the expression (percentage of positive cells and GMFI) of (A) gp130 D1 domain and (B) global gp130 was determined on monocytes by flow cytometry. Peripheral blood monocyte subsets were gated by FSC/SSC scatter properties. Representative histograms (right) of D1 domain and gp130 expression on treated or untreated (control) cells are shown. The data (mean±SEM) are representative of two independent experiments performed in quadruplicate.

Article Snippet: For detection of gp130 (CD130) expression and D1 domain expression, stimulated PBMC were harvested, fixed and stained with the following antibodies: mouse monoclonal anti-human gp130 (clone: B-T2, Abcam) followed by PE-labeled monoclonal anti-mouse IgG1 (Biolegend), together with Alexa fluor-647 conjugated polyclonal anti-human gp130 antibody (eBioscience) in concentrations recommended by the manufacturer.

Techniques: Activity Assay, Expressing, Incubation, Cytometry, Control

Journal: Biochimica et biophysica acta

Article Title: Trypanosoma cruzi, the causative agent of Chagas disease, modulates interleukin-6-induced STAT3 phosphorylation via gp130 cleavage in different host cells.

doi: 10.1016/j.bbadis.2012.12.003

Figure Lengend Snippet: Fig. 6. Cruzipain activity reduces the gp130 D1 domain expression on human peripheral blood lymphocytes. Mononuclear cells were incubated with active cruzipain for 120 min. Then, the expression (percentage of positive cells and GMFI) of (A) gp130 D1 Domain and (B) global gp130 was determined on lymphocytes by flow cytometry. Peripheral blood lymphocyte subsets were gated by FSC/SSC scatter properties. Representative histograms (right) of D1 domain and gp130 expression on treated or untreated (control) cells are shown. The data (mean±SEM) are representative of two independent experiments performed in quadruplicate.

Article Snippet: For detection of gp130 (CD130) expression and D1 domain expression, stimulated PBMC were harvested, fixed and stained with the following antibodies: mouse monoclonal anti-human gp130 (clone: B-T2, Abcam) followed by PE-labeled monoclonal anti-mouse IgG1 (Biolegend), together with Alexa fluor-647 conjugated polyclonal anti-human gp130 antibody (eBioscience) in concentrations recommended by the manufacturer.

Techniques: Activity Assay, Expressing, Incubation, Cytometry, Control

Journal: Biochimica et biophysica acta

Article Title: Trypanosoma cruzi, the causative agent of Chagas disease, modulates interleukin-6-induced STAT3 phosphorylation via gp130 cleavage in different host cells.

doi: 10.1016/j.bbadis.2012.12.003

Figure Lengend Snippet: Fig. 8. Recombinant human gp130 is cleaved by cruzipain enzymatic activity. Recombi- nant gp130 ectodomain was incubated with active cruzipain for 120 min at 37 °C (lane 2) or maintained in PBS (lane 1). Samples were separated on SDS-PAGE, electrotransferred to the nitrocellulose membrane and the proteins were evidenced by silver staining. Cruzipain, at the concentration employed in the experiment, was run on lane 3.

Article Snippet: For detection of gp130 (CD130) expression and D1 domain expression, stimulated PBMC were harvested, fixed and stained with the following antibodies: mouse monoclonal anti-human gp130 (clone: B-T2, Abcam) followed by PE-labeled monoclonal anti-mouse IgG1 (Biolegend), together with Alexa fluor-647 conjugated polyclonal anti-human gp130 antibody (eBioscience) in concentrations recommended by the manufacturer.

Techniques: Recombinant, Activity Assay, Incubation, SDS Page, Membrane, Silver Staining, Concentration Assay