human dll4 his tag  (Creative BioMart)

Journal: The FEBS journal

Article Title: Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

doi: 10.1111/febs.16946

Figure Lengend Snippet: Fig. 2. Loss of Dmt1 results in diminished endogenous Notch1 signalling, which is rescued by overexpression of Nicd. (A) mRNA expression levels of Notch target genes, Hey1 and Hey2, in Dmt1 WT and KO MEFs stimulated with Dll4 and treated with DMSO or GSI for 24 h, measured by qRT-PCR. Csnk2a2 mRNA expression was employed as a housekeeping control [one-way ANOVA (Tukey compari- son), *P < 0.05, **P < 0.01, ***P < 0.001, significantly compared with Dmt1 WT cells stimulated with Dll4]. (B) Fluorescent ectopic expres- sion of Nicd1-mCherryGFP in Dmt1 WT and KO cells upon doxycycline (DOX)-induction. Cells were counterstained with DAPI. Scale bar: 10 lm. GSI, c-secretase inhibitor dibenzazepine. Data are representative of three independent experiments, and values are expressed in mean SD.

Article Snippet: Delta-like 4 (Dll4) stimulation was performed using recombinant human Dll4 (R&D Systems, Minneapolis, MN, USA; cat. #1506-D4-050/CF).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Bmp9 regulates Notch signaling and the temporal dynamics of angiogenesis via Lunatic Fringe

doi: 10.1101/2023.09.25.557123

Figure Lengend Snippet: Bmp9 upregulates LFng expression in endothelial cells. (A) Schematic representation of the crosstalk between Bmp9 and Vegf-Notch signaling in ECs, as well as of the simulation and in vitro experiments. Green and blue lines represent previously established characteristics of the crosstalk; in red, the hypothesized LFng upregulation by Bmp9 is reported. (B-E) Experiments (full color) and simulations (striped color) of qPCR analysis of Hes1, Hey1 and LFng in HUVECs, induced by Bmp9 injection and/or Dll4 coating after 24h stimulation, with (bright color) or without (shaded color) LFng siRNA treatment. Number of repeats was higher than 4 in all cases ((B, D) N=5; (E) N=4). (F) Western blot analysis of NICD and LFng compared to the safe-keeping gene beta-actin in HUVECs with control (left) or LFng siRNA treatment (right) upon stimulation with 10ng/ml Bmp9, up to 24h, with relative quantification normalized over unstimulated data (bottom). The number of independent repeats per condition was N = 3. (G) IsolectinB4 and LFng staining of wild type and Alk1ΔEC retinal vessels, with associated normalized quantification of qPCR analysis. For the qPCR analysis, the number of independent experimental repeats per condition was N = 3. Scale bar, 200 µm. All values are mean +/− SD. *p < 0.05; **p<0.01; ***p<0.001; ****p<0.0001; (B, D-E) ANOVA, (G) Unpaired t-test.

Article Snippet: Quantitect qPCR primers for Jag1 (QT00031948), Hey1 (QT00035644), Hey2 (QT00026971), Dll4 (QT00081004), Hes1 (QT00039648), LFng (QT00212240), MFng (QT00084007), and RFng (QT01160075) were purchased from Qiagen, Recombinant BMP9 (3209-BP-010) and Dll4 (1506-D4-050) were obtained from R&D systems.

Techniques: Expressing, In Vitro, Injection, Western Blot, Control, Quantitative Proteomics, Staining

Journal: bioRxiv

Article Title: Bmp9 regulates Notch signaling and the temporal dynamics of angiogenesis via Lunatic Fringe

doi: 10.1101/2023.09.25.557123

Figure Lengend Snippet: Bmp9-mediated LFng upregulation potentiates EC-pericyte interactions. (A) Schematic representation of the signaling crosstalk between ECs and pericytes, as well as of the simulation and in vitro experiments. Full names correspond to unbound ligands and receptors (e.g. Notch1), while capital letters with numbers correspond to bound receptors (e.g. N1.D4 is the bound Notch1-Dll4 complex). Straight and curved arrows represent upregulation and protein complex formation, respectively, while the ⊣ symbol represents inhibition. (B-G) Experiments (full color) and simulations (striped color) of qPCR analysis of Hes1 and Hey1 in HUVECs and pericytes, cultured alone or in co-culture, induced by Bmp9 injection (10 ng/ml) after 24h stimulation, with (bright color) or without (shaded color) Jag1 or LFng siRNA treatment. The number of independent experimental repeats was N = 5. (H) qPcR analysis of LFng and Jag1 in HUVECs induced by Bmp9 injection (10 ng/ml) after 24h stimulation, with or without LFng siRNA treatment. The number of independent experimental repeats was N = 7. (I) qPCR analysis of Jag1 in HUVECs stimulated by Bmp9 (10 ng/ml) and/or Dll4 (10 ng/ml) at different time points, up to 24h. The number of independent experimental repeats was N=7. All values are mean +/− SD. *p < 0.05; **p<0.01; ***p<0.001; ****p<0.0001; ANOVA.

Article Snippet: Quantitect qPCR primers for Jag1 (QT00031948), Hey1 (QT00035644), Hey2 (QT00026971), Dll4 (QT00081004), Hes1 (QT00039648), LFng (QT00212240), MFng (QT00084007), and RFng (QT01160075) were purchased from Qiagen, Recombinant BMP9 (3209-BP-010) and Dll4 (1506-D4-050) were obtained from R&D systems.

Techniques: In Vitro, Inhibition, Cell Culture, Co-Culture Assay, Injection

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Expression fold change of EREG when THP-1 monocytes were incubated with each indicated cytokine. (B) EREG protein quantification from supernatant of THP-1 incubated with IFNa2 for 4 hours, n=4 per group, data is representative from 2 independent experiments. (C-E) EREG expression fold change from freshly isolated peripheral blood CD14+ monocytes (C) and CD1c+ dendritic cell precursors (D) or cultured human BMDC (E) after incubation with IFNα2. (F) Expression fold change of NOTCH ligands, receptors, and target genes by HFFs incubated with recombinant human EREG (n=5). (G) HES1 expression fold change in SSc fibroblasts after incubation with EREG for 4 hours, n=5 per group. (H) EREG relative expression by BMDC primed with IFNα2 prior to exposure to NOTCH ligand DLL4 (n=3–4 per time point in each group). Statistics compare each group ± DLL4. (I) Relative expression of EGFR ligands by HFF (n=3). Genes with fewer than three points were below detectable level. (J) Changes in ECM gene expression when HFF were incubated with media alone (NT) or EREG neutralizing antibody (Ereg Ab). FNEDA refers to the extra domain A-containing isoform of fibronectin (n=5 per group). (K) Model of EREG-NOTCH circuit between monocyte-derived DC3 and fibroblasts. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (A-H, J) and one-way ANOVA with Tukey multiple-comparisons test (I).

Article Snippet: To test the effects of NOTCH ligands, monocytes were incubated in media alone or IFNa2 1000 U/mL for 6 hours at 37° C. During the last 45 minutes, recombinant NOTCH ligands DLL4 (R&D Biosciences 1506-D4) and NOV (R&D Biosciences 1640-NV) each 10 μg/mL were adhered to the bottom of 48 well plates at 37° C for 45 minutes as described ( 90 ).

Techniques: Expressing, Incubation, Isolation, Cell Culture, Recombinant, Derivative Assay, Two Tailed Test