anti sdc4 apc  (R&D Systems)

Journal: bioRxiv

Article Title: Inhibition of MMP 2&9 protects the endothelial glycocalyx and improves diastolic function in diabetic cardiomyopathy

doi: 10.1101/2023.12.18.572183

Figure Lengend Snippet: Cells were pre-incubated with SB-3CT for 2 hours before being treated with TNF-α for 6 hours. (a) SDC4 cell surface expression. Fluorescence intensity was normalized to cell number (n=5 for all groups; control vs TNF-α, *p<0.05; TNF-α vs TNF-α + SB-3CT, **p<0.01; One-way ANOVA). (b) SDC4 mRNA expression (n=5 for all groups; Control vs TNF-α, ****p<0.0001; TNF-α vs TNF-α + SB-3CT, *P<0.05; One-way ANOVA). (c) SDC4 concentration in the conditioned media (n=4 for all groups; Control vs TNF-α, ***p<0.001; TNF-α vs TNF-α + SB-3CT, **p<0.01; One-way ANOVA). (d) GAG concentration in the conditioned media (n=4 for all groups; Control vs TNF-α, *p<0.05; TNF-α vs TNF-α + SB-3CT, **p<0.01; One-way ANOVA)

Article Snippet: The cells were incubated with an anti-SDC4 primary antibody at 10 mg/ml in blocking buffer (AF2918-SP, Bio-Techne) for 1h at room temperature.

Techniques: Incubation, Expressing, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Inhibition of MMP 2&9 protects the endothelial glycocalyx and improves diastolic function in diabetic cardiomyopathy

doi: 10.1101/2023.12.18.572183

Figure Lengend Snippet: (a) MMP9 mRNA expression (n=5 for both groups; *p<0.05; unpaired t test). (b) MMP9 activity in the conditioned media (n=5 for all groups; control vs TNF-α, *p<0.05; TNF-α vs TNF-α + SB-3CT, **p< 0.01; One-way ANOVA). (c) MMP9 mRNA expression from shRNA MMP9 cells (n=4 for both groups; **p<0.01; unpaired t test). (d) SDC4 mRNA expression (n=4 for all groups; scrambled control vs scrambled control + TNF-α, ****p<0.0001. Scrambled control + TNF-α vs shRNA MMP9 + TNF-α, **p<0.01; One-way ANOVA). No significant difference found between shRNA MMP9 and scrambled control (p=0.5). (e) SDC4 concentration in the conditioned media (n=4 for all groups; scrambled control vs scrambled control + TNF-α, ***p<0.001. Scrambled control + TNF-α vs shRNA MMP9 + TNF-α, *p<0.05)

Article Snippet: The cells were incubated with an anti-SDC4 primary antibody at 10 mg/ml in blocking buffer (AF2918-SP, Bio-Techne) for 1h at room temperature.

Techniques: Expressing, Activity Assay, shRNA, Concentration Assay

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 4. Effect of SDC4 knockdown (KD) or heparin inhibition on virus internalization into Calu-3 cells. SDC4 KD in Calu-3 cells was performed previously using a lentiviral vector specific to human SDC4. (A) SDC4 expression levels were measured with imaging flow cytometry, as shown by the representative histograms and cellular images. Detected SDC4 levels of KD cells were normalized to WT Calu-3 cells as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05. (B,C) SDC4 KD and WT Calu-3 cells were exposed to 1 MOI of the heat-inactivated WT SCV2, Delta, and Omicron variants. For GAG inhibition, the viruses were preincubated with heparin (200 ug/mL for 30 min at 37 ◦C) before being added to the cells. (D,E) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of WT or SDC4 KD Calu-3 cells treated with the viruses in the presence or absence of heparin. Scale bar = 20 µm. (F,G) Detected intracellular fluorescent signals were normalized to WT Calu-3 (F) cells or cells untreated with heparin (G) as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: The samples were then immunoblotted onto PVDF membranes, and SDC4 proteins were detected with specific human SDC4 antibodies (5G9, Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-12766) and anti-mouse IgG-HRP secondary antibody (Invitrogen, Carlsbad, CA, USA; cat. no. 31450).

Techniques: Knockdown, Inhibition, Virus, Plasmid Preparation, Expressing, Imaging, Cytometry

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 5. SDC4 binding of WT SCV2 and the Delta an Omicron variants. (A) SDS-PAGE showing SDC4 immunoprecipitated with an antibody specific for the spike’s amino acid sequence 1000-1200 from extracts of virus-treated Calu-3 cells. Lane 1: a total of 1 ug of recombinant SDC4; lanes 2–4: immunoprecipitates of Calu-3 cells treated with either WT SCV2, Delta, or Omicron, respectively; Lane 5: immunoprecipitate of untreated control Calu-3 cells. Standard protein size markers are indicated on the right. SDC4 signals were detected with UVITEC Alliance Q9 Advanced Imager, and the intensity of bands was analyzed with the NineAlliance© software. (B) Detected band intensities were normalized to WT SCV2-treated Calu-3 cells as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ns: not significant.

Article Snippet: The samples were then immunoblotted onto PVDF membranes, and SDC4 proteins were detected with specific human SDC4 antibodies (5G9, Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-12766) and anti-mouse IgG-HRP secondary antibody (Invitrogen, Carlsbad, CA, USA; cat. no. 31450).

Techniques: Binding Assay, SDS Page, Immunoprecipitation, Sequencing, Virus, Recombinant, Control, Software

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 6. Effect of SDC4 KD on the cellular entry and gene delivery of WT SCV2, Delta, or Omicron PSVs in Calu-3 cells. SDC4 KD and WT Calu-3 cells were treated with either WT SCV2, Delta, or Omicron PSVs. (A,B) Representative cellular images and flow cytometry histograms showing the intracellular fluorescence of WT or SDC4 KD Calu-3 cells treated with the PSVs. Scale bar = 20 µm. (C) Detected fluorescence intensities were normalized to WT Calu-3 cells treated with the respective PSVs. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: The samples were then immunoblotted onto PVDF membranes, and SDC4 proteins were detected with specific human SDC4 antibodies (5G9, Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-12766) and anti-mouse IgG-HRP secondary antibody (Invitrogen, Carlsbad, CA, USA; cat. no. 31450).

Techniques: Cytometry

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 4. Effect of SDC4 knockdown (KD) or heparin inhibition on virus internalization into Calu-3 cells. SDC4 KD in Calu-3 cells was performed previously using a lentiviral vector specific to human SDC4. (A) SDC4 expression levels were measured with imaging flow cytometry, as shown by the representative histograms and cellular images. Detected SDC4 levels of KD cells were normalized to WT Calu-3 cells as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05. (B,C) SDC4 KD and WT Calu-3 cells were exposed to 1 MOI of the heat-inactivated WT SCV2, Delta, and Omicron variants. For GAG inhibition, the viruses were preincubated with heparin (200 ug/mL for 30 min at 37 ◦C) before being added to the cells. (D,E) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of WT or SDC4 KD Calu-3 cells treated with the viruses in the presence or absence of heparin. Scale bar = 20 µm. (F,G) Detected intracellular fluorescent signals were normalized to WT Calu-3 (F) cells or cells untreated with heparin (G) as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis FlowSight, Luminex Corporation, Austin, TX, USA) with APCconjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A).

Techniques: Knockdown, Inhibition, Virus, Plasmid Preparation, Expressing, Imaging, Cytometry

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 5. SDC4 binding of WT SCV2 and the Delta an Omicron variants. (A) SDS-PAGE showing SDC4 immunoprecipitated with an antibody specific for the spike’s amino acid sequence 1000-1200 from extracts of virus-treated Calu-3 cells. Lane 1: a total of 1 ug of recombinant SDC4; lanes 2–4: immunoprecipitates of Calu-3 cells treated with either WT SCV2, Delta, or Omicron, respectively; Lane 5: immunoprecipitate of untreated control Calu-3 cells. Standard protein size markers are indicated on the right. SDC4 signals were detected with UVITEC Alliance Q9 Advanced Imager, and the intensity of bands was analyzed with the NineAlliance© software. (B) Detected band intensities were normalized to WT SCV2-treated Calu-3 cells as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ns: not significant.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis FlowSight, Luminex Corporation, Austin, TX, USA) with APCconjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A).

Techniques: Binding Assay, SDS Page, Immunoprecipitation, Sequencing, Virus, Recombinant, Control, Software

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 6. Effect of SDC4 KD on the cellular entry and gene delivery of WT SCV2, Delta, or Omicron PSVs in Calu-3 cells. SDC4 KD and WT Calu-3 cells were treated with either WT SCV2, Delta, or Omicron PSVs. (A,B) Representative cellular images and flow cytometry histograms showing the intracellular fluorescence of WT or SDC4 KD Calu-3 cells treated with the PSVs. Scale bar = 20 µm. (C) Detected fluorescence intensities were normalized to WT Calu-3 cells treated with the respective PSVs. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis FlowSight, Luminex Corporation, Austin, TX, USA) with APCconjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A).

Techniques: Cytometry