Journal: International Journal of Molecular Sciences

Article Title: Syndecan-3 as a Novel Biomarker in Alzheimer’s Disease

doi: 10.3390/ijms23063407

Figure Lengend Snippet: SDC3 expression of TNF-α-treated SH-SY5Y and hCMEC/D3 cells. Cells were incubated with or without (i.e., controls) 5 ng/mL TNF-α for 7 days. After incubation, the cells were treated with APC-labeled SDC3 antibody, and SDC3 was measured with imaging flow cytometry. ( A ) Representative flow cytometry histograms showing the SDC3 expression of SH-SY5Y and hCMEC/D3 cells. ( B ) Brightfield (BF) and fluorescent cellular images of SH-SY5Y and hCMEC/D3 cells treated with APC-labeled SDC3 antibody. Scale bar = 20 μm. ( C ) Detected fluorescence intensities were normalized control cells untreated with TNF-α. The bars represent the mean + SEM of ten independent experiments. Statistical significance vs. controls was assessed with ANOVA. *** p < 0.001.

Article Snippet: SDC3 expression of SH-SY5Y and hCMEC/D3 cells incubated with or without 5 ng/mL of recombinant TNF-α (cat. no. 210-TA-100/CF, RnD Systems, Minneapolis, MN, USA) for 7 days was measured with an AMNIS FlowSight imaging flow cytometer (Luminex Corporation, Austin, TX, USA) by using APC-labeled anti-human SDC3 antibody (polyclonal goat IgG, cat. no. FAB3539A, RnD Systems) as described previously [ , ].

Techniques: Expressing, Incubation, Labeling, Imaging, Flow Cytometry, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-3 as a Novel Biomarker in Alzheimer’s Disease

doi: 10.3390/ijms23063407

Figure Lengend Snippet: APPSWE-Tau mice exhibits increased TNF-α concentrations in the brain and the blood. ( A , B ) TNF-α concentrations of brain extracts ( A ) and whole blood ( B ) of APPSWE-Tau mice, along with representative WT controls, were measured with a mouse SDC3 ELISA kit. Each group contained 8 animals. The bars represent the mean + SEM. Statistical significance vs. WT was assessed with ANOVA. ** p < 0.01. ( C ) Linear regression between the TNF-α content of blood and brain.

Article Snippet: SDC3 expression of SH-SY5Y and hCMEC/D3 cells incubated with or without 5 ng/mL of recombinant TNF-α (cat. no. 210-TA-100/CF, RnD Systems, Minneapolis, MN, USA) for 7 days was measured with an AMNIS FlowSight imaging flow cytometer (Luminex Corporation, Austin, TX, USA) by using APC-labeled anti-human SDC3 antibody (polyclonal goat IgG, cat. no. FAB3539A, RnD Systems) as described previously [ , ].

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-3 as a Novel Biomarker in Alzheimer’s Disease

doi: 10.3390/ijms23063407

Figure Lengend Snippet: APPSWE-Tau mice exhibits increased SDC3 concentrations in the brain and the liver. ( A , B ) SDC3 concentrations of the brain ( A ) and liver ( C ) extracts of APPSWE-Tau mice and representative WT controls were measured with ELISA. Each group contained 8 animals. The bars represent the mean + SEM. Statistical significance vs. WT was assessed with ANOVA. * p < 0.05. ( C , D ) Linear regression between the TNF-α and SDC3 concentrations in the brain ( C ) and liver ( D ).

Article Snippet: SDC3 expression of SH-SY5Y and hCMEC/D3 cells incubated with or without 5 ng/mL of recombinant TNF-α (cat. no. 210-TA-100/CF, RnD Systems, Minneapolis, MN, USA) for 7 days was measured with an AMNIS FlowSight imaging flow cytometer (Luminex Corporation, Austin, TX, USA) by using APC-labeled anti-human SDC3 antibody (polyclonal goat IgG, cat. no. FAB3539A, RnD Systems) as described previously [ , ].

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-3 as a Novel Biomarker in Alzheimer’s Disease

doi: 10.3390/ijms23063407

Figure Lengend Snippet: PBECs of APPSWE-Tau mice exhibits increased SDC3 expression. Isolated PBECs were treated with PECAM-1 and SDC3 antibodies, and SDC3 expression of PECAM-1 positive cells was analyzed with imaging flow cytometry. ( A ) Representative histogram showing the SDC3 expression of PBECs isolated from APPSWE-Tau and WT mice. ( B ) BF and fluorescent cellular images of PECAM-1 and SDC3 antibody-treated PBECs, isolated from APPSWE-Tau and WT mice. Each group contained eight animals. SDC3 expression of each sample was measured twice. ( C ) Detected fluorescence intensities were normalized to WT. The bars represent the mean + SEM. Statistical significance vs. WT was assessed with ANOVA. * p < 0.05. ( D ) Linear regression between in PBECs’ relative SDC3 expression and blood TNF-α concentrations.

Article Snippet: SDC3 expression of SH-SY5Y and hCMEC/D3 cells incubated with or without 5 ng/mL of recombinant TNF-α (cat. no. 210-TA-100/CF, RnD Systems, Minneapolis, MN, USA) for 7 days was measured with an AMNIS FlowSight imaging flow cytometer (Luminex Corporation, Austin, TX, USA) by using APC-labeled anti-human SDC3 antibody (polyclonal goat IgG, cat. no. FAB3539A, RnD Systems) as described previously [ , ].

Techniques: Expressing, Isolation, Imaging, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-3 as a Novel Biomarker in Alzheimer’s Disease

doi: 10.3390/ijms23063407

Figure Lengend Snippet: Monocytes isolated from APPSWE-Tau mice exhibits increased SDC3 expression. Isolated monocytes were treated with CD11b and SDC3 antibodies, and SDC3 expression of CD11b positive cells was analyzed with imaging flow cytometry. ( A ) Representative histogram showing the SDC3 expression of monocytes isolated from APPSWE-Tau and WT mice. ( B ) BF and fluorescent cellular images of CD11b and SDC3 antibody-treated monocytes, isolated from APPSWE-Tau and WT mice. Each group contained eight animals. SDC3 expression of each sample was measured twice. ( C ) Detected fluorescence intensities were normalized to WT. The bars represent the mean + SEM. Statistical significance vs. WT was assessed with ANOVA. *** p < 0.001. ( D ) Linear regression between the SDC3 expression of monocytes and Aβ plaque loads.

Article Snippet: SDC3 expression of SH-SY5Y and hCMEC/D3 cells incubated with or without 5 ng/mL of recombinant TNF-α (cat. no. 210-TA-100/CF, RnD Systems, Minneapolis, MN, USA) for 7 days was measured with an AMNIS FlowSight imaging flow cytometer (Luminex Corporation, Austin, TX, USA) by using APC-labeled anti-human SDC3 antibody (polyclonal goat IgG, cat. no. FAB3539A, RnD Systems) as described previously [ , ].

Techniques: Isolation, Expressing, Imaging, Flow Cytometry, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syndecan-3 positively regulates the pro-inflammatory function of macrophages

doi: 10.1007/s00018-025-05649-1

Figure Lengend Snippet: SDC3, but not the other syndecans, is upregulated in response to IFNγ in human macrophages. ( a ) SDC1 , SDC2 , SDC3 and SDC4 mRNA levels in control and IFNγ-treated THP-1 macrophages, as assessed by RT-qPCR ( n = 4–6). ( b ) Representative flow histograms (upper panel) and pooled data (lower panel) showing the protein expression of SDC1, SDC2, SDC3 and SDC4 in control and IFNγ-treated THP-1 macrophages ( n = 4–6). ( c ) Representative confocal images ( n = 3) of control, IFNγ- or IL-4/IL-13-treated THP-1 macrophages immunostained for DAPI and SDC3 (scale bar 5 μm). ( d ) SDC3 mRNA levels in control, IFNγ- and IL-4/IL-13-treated THP-1 macrophages, as assessed by RT-qPCR ( n = 3). Means ± SEM. Statistically significant difference from controls or between indicated groups is shown by ns: not significant, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Article Snippet: SDC3 primary antibody (R&D Systems Cat. #: AF3539), was incubated overnight at 4 °C in blocking buffer.

Techniques: Control, Quantitative RT-PCR, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syndecan-3 positively regulates the pro-inflammatory function of macrophages

doi: 10.1007/s00018-025-05649-1

Figure Lengend Snippet: SDC3 deficient macrophages exhibit aberrant proliferation, adhesion and expression of cell surface markers. ( a ) Representative immunoblots ( n = 3) of WT or SDC3 KO THP-1 macrophages stimulated with either 100 ng/ml of IFNγ or 20 ng/ml of IL-4/IL-13 cocktail. ( b ) SDC3 mRNA levels in control, IFNγ- and IL-4/IL-13-treated WT or SDC3 KO THP-1 macrophages, as assessed by RT-qPCR ( n = 3) ( c ) Representative brightfield images of WT or SDC3 KO THP-1 macrophages (scale bar 20 μm, n = 3). ( d ) Proliferation of WT or SDC3 KO THP-1 cells up to 5 days ( n = 3). ( e ) Adhesion activity of WT or SDC3 KO THP-1 macrophages, as quantified by Crystal Violet ( n = 2–3). ( f ) Flow cytometry data showing the expression of CD40, CD86 and CD163 in WT or SDC3 KO THP-1 macrophages stimulated with 100 ng/ml of IFNγ ( n = 7). Means ± SEM. Statistically significant difference from controls or between indicated groups is shown by ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: SDC3 primary antibody (R&D Systems Cat. #: AF3539), was incubated overnight at 4 °C in blocking buffer.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Activity Assay, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syndecan-3 positively regulates the pro-inflammatory function of macrophages

doi: 10.1007/s00018-025-05649-1

Figure Lengend Snippet: SDC3 -defective macrophages show distinctive gene expression patterns. ( a ) Heatmap of differentially expressed genes in IFNγ-treated THP-1 WT and SDC3 KO macrophages ( n = 3, purple represents upregulation and green represents downregulation). ( b ) Differentially expressed pathways in IFNγ-treated THP-1 WT and SDC3 KO macrophages where blue represents pathways upregulated in SDC3 KO macrophages, and orange represents downregulated pathways in SDC3 KO macrophages ( n = 3). ( c ) TNF , IL10 , iNOS , PD-L1 , CD86 , and VEGFA mRNA levels in IFNγ-treated THP-1 WT and SDC3 KO macrophages, as assessed by RT-qPCR ( n = 3). Means ± SEM. Statistically significant difference from controls or between indicated groups is shown by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Article Snippet: SDC3 primary antibody (R&D Systems Cat. #: AF3539), was incubated overnight at 4 °C in blocking buffer.

Techniques: Gene Expression, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syndecan-3 positively regulates the pro-inflammatory function of macrophages

doi: 10.1007/s00018-025-05649-1

Figure Lengend Snippet: SDC3 promotes macrophage phagocytic capacity and inhibits tumour-spheroid formation. ( a ) Left: Representative images of IFNγ-stimulated WT or SDC3 KO THP-1 macrophages co-cultured with MDA-MB-231-GFP breast cancer cells. Images were taken with a Leica SP8 Lightning confocal microscope (scale bar 20.5 μm, n = 3). Right: Quantification of phagocytosis rate by flow cytometry normalized to WT, using the inhibitor of actin polymerization Cytochalasin D as control ( n = 3). ( b ) Left: Representative images of IFNγ-treated WT or SDC3 KO THP-1 macrophages forming spheroids with MDA-MB-231-GFP cells. Images were taken using Nikon Eclipse TD 100 microscope at the indicated times (scale bar 100 μm, white colour represents GFP + tumour cells). Right: Proliferation of MDA-MB-231-GFP + tumour cells in each condition normalized to MDA-MB-231 condition, calculated after acquiring single cell suspensions of spheroids by flow cytometry ( n = 3). Means ± SEM. Statistically significant difference from controls or between indicated groups is shown by * p < 0.05, ** p < 0.01 and **** p < 0.0001

Article Snippet: SDC3 primary antibody (R&D Systems Cat. #: AF3539), was incubated overnight at 4 °C in blocking buffer.

Techniques: Cell Culture, Microscopy, Flow Cytometry, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syndecan-3 positively regulates the pro-inflammatory function of macrophages

doi: 10.1007/s00018-025-05649-1

Figure Lengend Snippet: SDC3 plays a role in macrophage pro-inflammatory functions. ( a ) Left: Representative confocal microscopy images of WT or SDC3 KO THP-1 macrophages stimulated with 100 ng/ml of IFNγ and incubated with pHrodo™ Green S. aureus bioparticles. Images were taken using a Leica SP8 Lightning confocal microscope (scale bar 10 μm). Right: Flow cytometry quantification of S. aureus phagocytosis normalized to WT, using the inhibitor of actin polymerization Cytochalasin D as control ( n = 3). ( b ) Cytokine quantification in cell supernatants from WT or SDC3 KO THP-1 macrophages stimulated with 100 ng/ml of IFNγ, using the LEGENDplex™ HU Essential Immune Response Panel ( n = 5). ( c ) Quantification of T cell proliferation index using CellTrace CFSE staining ( n = 5) in CD8 + T cells co-cultured with IFNγ-treated WT or SDC3 KO THP-1 macrophages. ( d ) Quantification of T cell activation markers PD-1 and CD95 by flow cytometry in CD8 + T cells co-cultured with IFNγ-treated WT or SDC3 KO THP-1 macrophages ( n = 5). Means ± SEM. Statistically significant difference from controls or between indicated groups is shown by ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Article Snippet: SDC3 primary antibody (R&D Systems Cat. #: AF3539), was incubated overnight at 4 °C in blocking buffer.

Techniques: Confocal Microscopy, Incubation, Microscopy, Flow Cytometry, Control, Staining, Cell Culture, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syndecan-3 positively regulates the pro-inflammatory function of macrophages

doi: 10.1007/s00018-025-05649-1

Figure Lengend Snippet: SDC3 deficient macrophages regulate EC migration and tube formation. ( a ) HUVEC migration over the indicated times in response to conditioned media (CM) from WT or SDC3 KO THP-1 macrophages stimulated with 100 ng/ml of IFNγ. Serum-free media (SFM) was used as negative control ( n = 4). ( b ) Quantification of HUVEC migration (Cell Index) shown in (a) at 48 h ( n = 4). ( c ) Representative images of Matrigel-grown HUVECs treated with supernatants from WT or SDC3 KO THP-1 macrophages stimulated with 100 ng/ml of IFNγ. Tube formation was imaged using an Olympus IX-83 inverted microscope (scale bar 1 mm, n = 4). ( d ) Quantification of total tube number shown in (c) at 24 h, relative to WT untreated control ( n = 4). ( e ) VEGFA quantification by ELISA in the supernatants of WT or SDC3 KO THP-1 macrophages stimulated with 100 ng/ml of IFNγ ( n = 4). ( f ) Angiogenic factor quantification in cell supernatants from WT or SDC3 KO THP-1 macrophages stimulated with 100 ng/ml of IFNγ, using the LEGENDplex™ HU Angiogenesis Panel 1 ( n = 4). Means ± SEM. Statistically significant difference from controls or between indicated groups is shown by ns: not significant, * p < 0.05 and *** p < 0.001

Article Snippet: SDC3 primary antibody (R&D Systems Cat. #: AF3539), was incubated overnight at 4 °C in blocking buffer.

Techniques: Migration, Negative Control, Inverted Microscopy, Control, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syndecan-3 positively regulates the pro-inflammatory function of macrophages

doi: 10.1007/s00018-025-05649-1

Figure Lengend Snippet: Macrophage-derived SDC3 plays a role in the regulation of the TME. The plethora of cells present in the TME interact to promote or inhibit tumour progression and growth. SDC3 is expressed by macrophages in response to the pro-inflammatory cytokine IFNγ and regulates many aspects of the TME. Firstly, SDC3 in macrophages reduces tumour proliferation and is necessary for macrophage-mediated tumour-cell phagocytosis. The expression of SDC3 promotes a pro-inflammatory phenotype in macrophages that results in the secretion of cytokines such as MCP-1 and IL-10, while reducing the release of IL-8. Additionally, SDC3 is necessary for the acquisition of an effector phenotype by T cells, shown by an increased expression of PD-1 and CD95 on T cells. Finally, macrophage-derived SDC3 plays a role in the inhibition of endothelial cell migration and proliferation through a reduction in the secretion of angiogenic factors such as VEGFA, PECAM-1 and IL-8

Article Snippet: SDC3 primary antibody (R&D Systems Cat. #: AF3539), was incubated overnight at 4 °C in blocking buffer.

Techniques: Derivative Assay, Expressing, Inhibition, Migration

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular internalization and fibrillation of amyloid-β(1–42)

doi: 10.1038/s41598-018-37476-9

Figure Lengend Snippet: Colocalization of Aβ1–42 and SDCs. SDC transfectants or WT SH-SY5Y cells were treated with either FITC-Aβ1–42 or FITC-Trf for 18 h at 37 °C. After incubation, the cells were permeabilized and treated with the respective APC-labeled SDC antibody. Nuclei of cells were stained with DAPI and cellular uptake was then analyzed with CLSM. ( a , c ) CLSM images of SDC transfectants treated either FITC-Aβ1–42 ( a ) or FITC-Trf ( c ) and respective APC-labeled SDC antibody. Representative images of three independent experiments are shown. Scale bar = 5 μm. ( b , d ) Mander’s overlap coefficient (MOC) ± SEM for the overlap of SDCs with FITC-Aβ1–42 ( b ) or FITC-Trf ( d ) was calculated by analysis of 21 images with ~7 cells in each image (from three separate samples). ( e ) CLSM images of WT SH-SY5Y cells treated with FITC-Aβ1–42 or FITC-Trf, along with APC-labeled SDC3 antibody. Representative images of three independent experiments are shown. Scale bar = 10 μm. ( f ) Mander’s overlap coefficient (MOC) ± SEM for the overlap of SDC3 with FITC-Aβ1–42 or FITC-Trf was calculated by analysis of 21 images with ~7 cells in each image (from three separate samples)

Article Snippet: The supernatant were then transferred to new tubes and combined with 5 μg of the human SDC3 antibody (R&D Systems).

Techniques: Incubation, Labeling, Staining

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular internalization and fibrillation of amyloid-β(1–42)

doi: 10.1038/s41598-018-37476-9

Figure Lengend Snippet: Colocalization of SDCs, Aβ1–42 and flotillins. SDC transfectants and WT SH-SY5Y cells were treated with 5-TAMRA-labeled Aβ1–42 for 18 h at 37 °C. After incubation, the cells were permeabilized and treated with the respective APC-labeled SDC antibody, along with either flotillin 1 (FLOT1) or flotillin 2 (FLOT2) antibodies (both Alexa Fluor 488-labeled). Nuclei of cells were stained with DAPI and cellular uptake was then analyzed with CLSM. Representative images of three independent experiments are shown. ( a , c ) CLSM images of SDC transfectants treated with FITC-Aβ1–42, one of the anti-flotillin antibodies and a respective APC-labeled SDC antibody. ( b , d ) Mander’s overlap coefficient (MOC) ± SEM for the overlap of SDCs with either FLOT1 or FLOT2 was calculated by analysis of 21 images with ~7 cells in each image (from three separate samples). ( e ) CLSM images of WT SH-SY5Y cells treated with 5-TAMRA-labeled Aβ1–42, APC-labeled SDC3 antibody and FLOT1 or FLOT2 antibodies. Scale bar = 10 μm. ( f ) Mander’s overlap coefficient (MOC) ± SEM for the overlap of SDC3 with either FLOT1 or FLOT2 was calculated by analysis of 21 images with ~7 cells in each image (from three separate samples).

Article Snippet: The supernatant were then transferred to new tubes and combined with 5 μg of the human SDC3 antibody (R&D Systems).

Techniques: Labeling, Incubation, Staining

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular internalization and fibrillation of amyloid-β(1–42)

doi: 10.1038/s41598-018-37476-9

Figure Lengend Snippet: Effect of undersulfation on Aß1–42 uptake and binding. WT K562 cells and SDC transfectants were incubated with sodium chlorate (NaClO3) prior to Aβ1–42 treatment. Effect of NaClO3 on HS expression, along with Aβ1–42 and attachment were assessed with flow cytometry. ( a ) Flow cytometry histograms showing HS expression of WT K562 cells or SDC transfectants after NaClO3 treatment. ( b ) Flow cytometry histograms representing intracellular fluorescence of FITC-Aβ1–42-treated cells preincubated with or without NaClO3. ( c ) The effect of NaClO3 were expressed as percent inhibition, calculated with the following formula: [(X − Y)/X] × 100, where X is the fluorescence intensity obtained on cells treated with Aβ1–42 in the absence of the NaClO3 and Y is the fluorescence intensity obtained on cells treated with either Aβ1–42 in the presence of the NaClO3. The bars represent mean ± SEM of four independent experiments. Statistical significance vs controls untreated with NaClO3 was assessed by analysis of variance (ANOVA). *p < 0.05 vs standards; **p < 0.01 vs standards; ***p < 0.001 vs standards. ( d ) Scanning electron microscope visualization of cellular surface of Aβ1–42-treated WT K562 cells and SDC3, SDC4 transfectants, preincubated with or without NaClO3. Representative images of three independent experiments are shown. Scale bar = 1 μm.

Article Snippet: The supernatant were then transferred to new tubes and combined with 5 μg of the human SDC3 antibody (R&D Systems).

Techniques: Binding Assay, Incubation, Expressing, Flow Cytometry, Fluorescence, Inhibition, Microscopy

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular internalization and fibrillation of amyloid-β(1–42)

doi: 10.1038/s41598-018-37476-9

Figure Lengend Snippet: Effect of SDC3 on Aβ1–42 uptake and fibrillation in neurons. SDC3 transfectants created in SH-SY5Y cells were selected by measuring SDC3 expression with flow cytometry using APC-labeled anti-SDC3 antibody. HS expression of SDC3 transfectants, along with WT SH-SY5Y, was also measured with flow cytometry using anti-HS antibody. SDC3 transfectants and WT SH-SY5Y cells were treated with Aβ1–42 (with or without FITC label) at a concentration of 5 μM at 37 °C. Cells incubated with Aβ1–42 for 18 h were then processed for uptake, fibrillation studies and scanning electron microscopy. ( a ) Flow cytometry histograms representing SDC3, HS expression levels and intracellular fluorescence of Aβ1–42-treated WT SH-SY5Y cells and SDC3 transfectants. ( b ) Fold change in SDC3 and HS expression, along with Aβ1–42 uptake and fibrillation following SDC3 overexpression. The bars represent mean ± SEM of six independent experiments. Statistical significance vs Aβ1–42-treated WT SH-SY5Y cells (standards) was assessed by analysis of variance (ANOVA). *p < 0.05 vs Aβ1–42-treated WT SH-SY5Y cells (standards). ( c , d ) Linear regression between HS or SDC3 expression and Aβ1–42 uptake or fibrillation. ( e ) Scanning electron microscopy visualization of Aβ1–42 attachment and fibrillation on WT SH-SY5Y cells and SDC3 mutants. Representative images of three independent experiments are shown. Scale bar = 2 μm.

Article Snippet: The supernatant were then transferred to new tubes and combined with 5 μg of the human SDC3 antibody (R&D Systems).

Techniques: Expressing, Flow Cytometry, Labeling, Concentration Assay, Incubation, Electron Microscopy, Fluorescence, Over Expression

Journal: Journal of Molecular Endocrinology

Article Title: Syndecans modulate ghrelin receptor signaling

doi: 10.1530/JME-24-0070

Figure Lengend Snippet: Syndecans (SDCs) impair β-arrestin2 recruitment at growth hormone secretagogue receptor (GHSR). (A) Effect of SDC1 on ghrelin-stimulated β-arrestin2–GHSR interaction in HEK293 cells. Values were corrected for Emax of the control (*** P < 0.0005 versus 1:0 ratio GHSR:SDC1; # P < 0.05, ## P < 0.005 versus 1:2 ratio GHSR:SDC1). (B) Effect of SDC1 on the kinetic profile of the β-arrestin2–GHSR interaction upon ghrelin stimulation (outcome of RM-ANOVA shown on graph: P T , effect of time; P Ratio , effect of GHSR:SDC ratio; P TxRatio , interaction. * P < 0.05, all other ratios versus 1:0 ratio GHSR:SDC1; # P < 0.05, 1:1 and 1:2 versus 1:0 ratio; $ P < 0.05, 1:2 versus 1:0 ratio). Data derived from curve fitting of the kinetic profiles for β-arrestin2 recruitment by GHSR in cells transfected with control or increasing amounts of SDC1 expression plasmid. (C) Initial rate of recruitment. (D) Magnitude of peak response (letters a and b represent groups that are significantly different from each other ( P < 0.05; one-way ANOVA). Dose–response curve (E) (*** P < 0.0005 versus control; # P < 0.05 versus 1:2 ratio GHSR:SDC1) and kinetic curve (F) of the effect of all SDCs (GHSR:SDC of 1:2) on β-arrestin2 recruitment at GHSR (outcome of RM-ANOVA shown on graph. * P < 0.05, *** P < 0.001, all SDCs versus control; # P < 0.05, SDC1, SDC2 and SDC4 versus control; $ P < 0.05, SDC2 and SDC4 versus control. P T , effect of time; P Ratio , effect of GHSR:SDC ratio; P TxRatio , interaction). Data derived from curve fitting of the kinetic profiles for β-arrestin2 recruitment by GHSR in cells transfected with control or SDC1, SDC2, SDC3 or SDC4 expression plasmids. (G) Initial rate of recruitment. (H) Magnitude of peak response (letters a and b represent groups that are significantly different from each other ( P < 0.05; one-way ANOVA). Data are presented as mean ± SEM of three independent experiments. Emax, maximum response.

Article Snippet: Because the SinoBiological SDC3 expression plasmid contained a 5′ truncated cDNA variant (SinoBiological, Germany; HG12158-UT; NCBI, BC013974), we generated a full-length pCMV3-SDC3 expression construct by inserting the correct 5′ region from a GenScript plasmid (GenScript, The Netherlands; Clone ID Ohu18173; NM_014654.4) utilizing Hind III to Kfl I restriction sites.

Techniques: Control, Derivative Assay, Transfection, Expressing, Plasmid Preparation