Journal: Alzheimer's & Dementia

Article Title: Cranial bone maneuver ameliorates Alzheimer's disease pathology via enhancing meningeal lymphatic drainage function

doi: 10.1002/alz.14518

Figure Lengend Snippet: The effects of IL‐6 on LECs. (A and B) Cell viability of human dermal LECs exposed to IL‐6 for 24 and 48 h was measured using the CCK‐8 assay. The changes of mRNA levels of IL‐1β (C), IL‐6 (D), CCL2 (E), and TGF‐β (F) in human dermal LECs after IL‐6 treatment for 24 and 48 h. For tube‐formation assay (G), IL‐6 significantly increased the total length (H) and number of branches (I) of LECs, whereas Tocilizumab blocked such enhancement effect. For cell scratch assay (J), IL‐6 promotes the migration of LECs with significant increase of wound closure percentage after 12 (K) and 24 h (L), whereas Tocilizumab blocked such enhancement. * p < 0.05, ** p < 0.01, and *** p < 0.001, one‐way ANOVA with Bonferroni's post hoc tests among groups. ANOVA, analysis of variance; CBM, cranial bone maneuver; LECs, lymphatic endothelial cells.

Article Snippet: Then 5 × 10 4 cells were added per well with the treatment of recombinant human IL‐6 protein (20 ng/mL, 206‐IL‐025/CF, R&D Systems, Inc.), Tocilizumab (100 ng/mL, Anti‐Human IL‐6R, Humanized Antibody, HY‐P9917, MedChemExpress), or vehicle, and were incubated for 16 h at 37°C with 5% CO 2 .

Techniques: CCK-8 Assay, Tube Formation Assay, Wound Healing Assay, Migration

Journal:

Article Title: The Phenotypic Plasticity of Myeloma Plasma Cells as Expressed by Dedifferentiation into an Immature, Resilient, and Apoptosis-Resistant Phenotype

doi: 10.1158/1078-0432.CCR-05-0523

Figure Lengend Snippet: A–B, blocking IL-6 activity did not reverse the immature phenotype of cocultured myeloma cells. CD45/38 flow cytometry of multiple myeloma plasma cells from two patients before culture (precultured), after 6 weeks in culture with osteoclasts (cocultured), and after 4 additional weeks in culture with osteoclasts, anti-IL6, and anti-IL6R. Note an up-regulation of CD45 expression by osteoclasts that was not reversed by inhibition of IL-6 activity.

Article Snippet: Neutralizing polyclonal antibodies against human interleukin-6 (IL-6) and human IL-6 receptor (IL6R) were obtained from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Blocking Assay, Activity Assay, Flow Cytometry, Clinical Proteomics, Expressing, Inhibition

Journal: bioRxiv

Article Title: Enhancement of activation-induced T cell proliferation by SIRPG in a CD47-independent manner

doi: 10.1101/2025.05.01.651731

Figure Lengend Snippet: A . PBMC from healthy donors were stimulated with anti-CD3/anti-CD28 and restimulated with anti-CD3 on day 6. The surface level of SIRPG in CD4+ T cells was analyzed with FACS and shown in the overlay histograms (the left panel). The MFI of SIRPG from three independent donors over the time course is shown in the right panel. Adalimumab (ada), certolizumab (cer), etanercept (eta), tocilizumab (toc), or control IgG1 was added on day 0 during the stimulation. The MFI of SIRPG from non-blasting ( B ) and blasting ( C ) T cells is shown in the overlay histograms. The cumulative results from 3 independent experiments are shown in the corresponding bar graphs. D . PBMC were stimulated in the presence or absence of ada and the surface level of SIRPG was monitored with FACS at indicated time points. Representative data from one donor is shown in the overlay histograms and cumulated results from five donors are shown in the right panel. The data points from the same donors are connected with lines. E . PBMC were stimulated and ada or control IgG was added on day 3. The level of SIRPG was analyzed on day 6. Representative overlay histograms are shown in the left panel and the cumulative results from three donors are shown in the right panel. F . PBMC were treated with ada or control IgG without anti-CD3 stimulation. The surface level of SIRPG in T and non-T cells was analyzed on day 3. Representative overlay histograms are shown in the left panel and cumulative results from three donors are shown in the right panel.

Article Snippet: PBMCs were plated in 24-well plates (2-2.5 millions/1ml/well) pre-coated with anti-CD3 (2,5ug/ml, OKT3, Supplemental Table 3) and soluble anti-CD28 (2,0ug/ml, CD28.2, Supplemental Table 3) and stimulated with adalimumab (125ug/ml), etanercept (50ug/ml), certolizumab pegol (50ug/ml), tocilizumab (100ug/ml), or an isotype control anti-human IgG1 (125ug/ml, #BE0297, BioXCell, Lebanon, NH, USA) for indicated times before analyzing by flow cytometry.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: UVB-/Age-Dependent Upregulation of Inflammatory Factor Interleukin-6 Receptor (IL-6R) in Keratinocytes Stimulates Melanocyte Dendricity

doi: 10.3390/ijms262210971

Figure Lengend Snippet: Stimulation of melanocyte dendricity mediated by sIL-6R and upregulated expression of IL-6R in age spots. ( A ): In the co-culture of either young or adult keratinocytes, the length and the number of melanocyte dendrites were significantly increased in an age-dependent manner. ( B ): The increase in both dendrite length and number was significantly suppressed in the co-culture of Tocilizumab-treated keratinocytes compared with those in the control IgG-treated keratinocyte co-culture (Cont. IgG). ( C ): Representative fluorescent images of IL-6R protein expression in the epidermis of non-lesions and age spots ( n = 5 each). The relative level of IL-6R protein was significantly upregulated in age spots, being ~1.2-fold higher than that in the non-lesion area. FC, fold change. Dashed lines denote dermal–epidermal junction. DIC, differential interference contrast. Scale bars: 50 μm ( B , C ). The data are represented as means ± SDs. KC, keratinocyte.

Article Snippet: An IL-6R-specific neutralizing antibody, Tocilizumab (NBP2-75192, Novus Biologicals, Centennial, CO, USA), was used to evaluate the function of IL-6R secreted by keratinocytes.

Techniques: Expressing, Co-Culture Assay, Control

Journal: Journal of Biological Chemistry

Article Title: An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding

doi: 10.1074/jbc.m300456200

Figure Lengend Snippet: FIG. 1. Binding of endogenous ARTS-1 to IL-6R in NCI-H292 cells. A, co-immunoprecipitations (IP) were performed with either an anti-IL-6R monoclonal antibody directed against the IL-6R extracel- lular domain () or a murine IgG1 isotype control (IgG1) and immuno- blotted (IB) with either anti-ARTS-1 preimmune (PI) or immune () serum. B, reciprocal co-immunoprecipitations were performed with ei- ther anti-ARTS-1 preimmune or immune serum and immunoblotted with either an anti-IL-6R monoclonal antibody () or a murine IgG1 isotype control (IgG1).

Article Snippet: For immunoprecipitation, samples of membrane proteins (200 g) were incubated overnight, 4 °C, with 20 g of murine monoclonal anti-human IL-6R antibody against the IL-6R extracellular domain (R & D Systems) or 1 l of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 l of immobilized protein A/G beads (Pierce) for 2 h at room temperature.

Techniques: Binding Assay, Control

Journal: Journal of Biological Chemistry

Article Title: An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding

doi: 10.1074/jbc.m300456200

Figure Lengend Snippet: FIG. 2. ARTS-1 promotes IL-6R shedding. A, concentrations of sIL-6R in 24-h culture supernatants from two antisense (AS) and two sense (ARTS-1) cell lines were determined by ELISA (n 5). WT denotes wild type NCI-H292 cells. B, supernatants from ARTS-1 cell lines were concentrated 30-fold, subjected to SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti-IL-6R antibodies. C, effect of ARTS-1 on membrane-associated IL-6R in ARTS-1 cell lines. The amount of IL-6R present in 300 g of membrane proteins from ARTS-1 cell lines was determined by ELISA (n 5) and reported as pg of membrane-associated IL-6R (mIL-6R) per mg of membrane pro- tein. * p 0.05 versus mock-transfected cells. D, membrane fractions of ARTS-1 cell lines (40 g) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti-IL-6R antibodies.

Article Snippet: For immunoprecipitation, samples of membrane proteins (200 g) were incubated overnight, 4 °C, with 20 g of murine monoclonal anti-human IL-6R antibody against the IL-6R extracellular domain (R & D Systems) or 1 l of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 l of immobilized protein A/G beads (Pierce) for 2 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, SDS Page, Membrane, Transfection

Journal: Journal of Biological Chemistry

Article Title: An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding

doi: 10.1074/jbc.m300456200

Figure Lengend Snippet: FIG. 3. Generation and characterization of arts-1(/) cell lines. A, schematic representation of part of the arts-1 locus (top), the targeting vector (middle), and the targeted null locus (bottom). ARTS-1 contains a consensus zinc metalloprotease catalytic domain, HELAH(Y)18E, which is characteristic of aminopeptidase family members. Exon 6 encodes two zinc-binding sites (His-353 and His-357), whereas the first glutamic acid residue (Glu-354) is the active catalyst. The third zinc-binding site is encoded by the second glutamic acid (Glu-376) located in exon 7. The targeted null locus contains a substitution of a neomycin phosphotransferase cassette (Neo), in the antisense orientation, for arts-1 exons 5 and 6. A HindIII site is located in the intron between exons 3 and 4. Arrows indicate the locations of PCR primers. The locations of the arts-1 exon 15/17 primers are not shown. B, PCR amplification of genomic DNA (left panels) and RT-PCR amplification of mRNA (right panels) from wild type (WT) and three arts-1(/) cell lines. The IL-6R-TM primers amplify a 398-bp segment of IL-6R mRNA containing the transmembrane domain that is present in the proteolytically cleaved sIL-6R but is absent in differentially spliced sIL-6R.

Article Snippet: For immunoprecipitation, samples of membrane proteins (200 g) were incubated overnight, 4 °C, with 20 g of murine monoclonal anti-human IL-6R antibody against the IL-6R extracellular domain (R & D Systems) or 1 l of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 l of immobilized protein A/G beads (Pierce) for 2 h at room temperature.

Techniques: Plasmid Preparation, Binding Assay, Residue, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding

doi: 10.1074/jbc.m300456200

Figure Lengend Snippet: FIG. 4. Knock-out arts-1(/) cell lines do not shed IL-6R. A, duplicate samples (20 g) of membrane proteins from wild type (WT) NCI-H292 cells and three arts-1(/) clones were separated by SDS- PAGE, transferred to nitrocellulose membranes, and reacted with ARTS-1 or MUC1 antibodies. B, concentrations of sIL-6R in culture supernatants from wild type NCI-H292 cells and arts-1(/) cell lines, incubated for 24 h with or without 0.1 M PMA, were quantified by ELISA (n 5). *, p 0.05 arts-1(/) cell lines versus wild type NCI-H292 cells. **, p 0.05 PMA-treated wild type NCI-H292 cells versus untreated wild type NCI-H292 cells. ***, p 0.05 PMA-treated arts-1(/) cell lines versus PMA-treated wild type NCI-H292 cells.

Article Snippet: For immunoprecipitation, samples of membrane proteins (200 g) were incubated overnight, 4 °C, with 20 g of murine monoclonal anti-human IL-6R antibody against the IL-6R extracellular domain (R & D Systems) or 1 l of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 l of immobilized protein A/G beads (Pierce) for 2 h at room temperature.

Techniques: Knock-Out, Membrane, Clone Assay, SDS Page, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding

doi: 10.1074/jbc.m300456200

Figure Lengend Snippet: FIG. 6. TAPI inhibits ARTS-1-mediated increases in IL-6R shedding. ARTS-1 cell lines were incubated for 24 h with or without TAPI-0, TAPI-1, or TAPI-2 (25 M). Amounts of sIL-6R present in cell culture supernatants were quantified by ELISA (n 5). *, p 0.05 versus no TAPI.

Article Snippet: For immunoprecipitation, samples of membrane proteins (200 g) were incubated overnight, 4 °C, with 20 g of murine monoclonal anti-human IL-6R antibody against the IL-6R extracellular domain (R & D Systems) or 1 l of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 l of immobilized protein A/G beads (Pierce) for 2 h at room temperature.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding

doi: 10.1074/jbc.m300456200

Figure Lengend Snippet: FIG. 5. IL-6R shedding is reconsti- tuted by ARTS-1 expression in arts- 1(/) cell lines. A, duplicate samples (20 g) of membrane proteins from wild type NCI-H292 cells (WT) and arts-1(/) cell lines that were transiently transfected with plasmids encoding either full-length ARTS-1 or ARTS-1 catalytic site mutants (H353P, H357V, H353P, and E354V) were separated by SDS-PAGE, trans- ferred to nitrocellulose membranes, and reacted with ARTS-1 antibodies. No ARTS-1 protein was detected in cells transfected with empty pTarget mamma- lian expression vector (Mock). B, concen- trations of sIL-6R in 24-h culture super- natants from wild type NCI-H292 cells and transiently transfected arts-1(/) cell lines were quantified by ELISA (n 5). *, p 0.05 arts-1(/) cell lines transfected with full-length ARTS-1 versus mock- transfected cells.

Article Snippet: For immunoprecipitation, samples of membrane proteins (200 g) were incubated overnight, 4 °C, with 20 g of murine monoclonal anti-human IL-6R antibody against the IL-6R extracellular domain (R & D Systems) or 1 l of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 l of immobilized protein A/G beads (Pierce) for 2 h at room temperature.

Techniques: Expressing, Membrane, Transfection, SDS Page, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding

doi: 10.1074/jbc.m300456200

Figure Lengend Snippet: FIG. 7. GST-ARTS-1 does not cleave an IL-6R peptide substrate. A model IL-6R peptide substrate and purified GST-ARTS-1 were subjected to reverse phase HPLC utilizing a C18 column. Ab- sorbance at 214 nm is shown as a function of elution time. A 22-amino acid model IL-6R peptide substrate (RDSAN- ATSLPVQDSSSVPLPTF) that contains the IL-6R cleavage site (A) was synthe- sized to serve as a model substrate for GST-ARTS-1 (D). The GST-ARTS-1 fu- sion protein was catalytically active and had aminopeptidase activity against leucine, methionine, alanine, and phenyl- alanine p-nitroanilide model substrates. Both the expected N-terminal 12-amino acid (RDSANATSLPVQ) cleavage prod- uct (B) and the C-terminal 10-amino acid (DSSSVPLPTF) cleavage product (C) were synthesized to serve as standards. The IL-6R model peptide substrate was incubated with catalytically active GST- ARTS-1 fusion protein for 2 h at 37 °C. As shown in E, the catalytically active GST- ARTS-1 did not cleave the IL-6R model peptide substrate.

Article Snippet: For immunoprecipitation, samples of membrane proteins (200 g) were incubated overnight, 4 °C, with 20 g of murine monoclonal anti-human IL-6R antibody against the IL-6R extracellular domain (R & D Systems) or 1 l of rabbit anti-human ARTS-1 immune or pre-immune serum, followed by addition of 200 l of immobilized protein A/G beads (Pierce) for 2 h at room temperature.

Techniques: Purification, Activity Assay, Synthesized, Incubation