jg 050 jag2 fc r d systems  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

doi: 10.1016/j.jbc.2022.101833

Figure Lengend Snippet: Notch pathway regulates gene expression of basement membrane-related collagenous ECMs in PDPN-expressing SSC progenies. A , strategy for experiments involving the xenovascular model and LY-411575, a Notch pathway inhibitor. Prior to their coculture with HUVECs, PDPN-expressing SSC progenies were pretreated with LY-411575 in vitro . Isolated PDPN-expressing SSC progenies were subjected to ECM transcript analysis. B – F , LY-411575 suppresses the basement membrane ECM upregulation of PDPN-expressing SSC progenies during cell–cell interactions with HUVECs. In this experiment, we targeted the ECM genes Col4a1 ( B ), Col4a2 ( C ), Lama5 ( D ), Nid1 ( E ), and Nid2 ( F ), which were altered during coculture with HUVECs. G – L , RT-qPCR evaluating the expression of the genes encoding proteins present in vascular basement membrane-related ECMs under Notch stimulation with recombinant Notch ligands. ( G ) All recombinant Notch ligands upregulated Hes1 expression. The expression levels of collagenous or noncollagenous basement membrane ECMs genes: type IV collagen alpha-chains Col4a1 ( H ) and Col4a2 ( I ) laminin alpha-chains lama5 ( J ), and nidogen isoforms Nid1 ( K ) and Nid2 (L). ∗ p < 0.01 . ∗∗ p < 0.01 . ∗∗∗ p < 0.01 . ∗∗∗∗ p < 0.0001 . N.S. indicates nonsignificance differences. For panel B – F , comparison between two groups was performed by the Student’s t test (n = 5 per group). For panel G – L , multi-group comparison was performed by one-way ANOVA and Dunnett’s t test (n = 5 per group, control group: Fc). The error bars represent SEMs. ECM, extracellular matrix; HUVECs, human umbilical vein endothelial cells PDPN, podoplanin; RT-qPCR, reverse transcribed-quantitative PCR; SSC, skeletal stem cell.

Article Snippet: Recombinant Notch ligand-IgG Fc fusion proteins (JAG1-Fc, JAG2-Fc, DLL1-Fc, and DLL4-Fc) or recombinant human IgG1 Fc (Fc) were obtained from R&D Systems.

Techniques: Expressing, Membrane, In Vitro, Isolation, Quantitative RT-PCR, Recombinant, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Neurochemistry international

Article Title: Reciprocal communication between astrocytes and endothelial cells is required for astrocytic glutamate transporter 1 (GLT-1) expression

doi: 10.1016/j.neuint.2020.104787

Figure Lengend Snippet: RNA-seq transcriptome data from the Barres group showing expression of the four Notch ligands capable of Notch activation, Dll1, Dll4, Jag1, and Jag2 in mouse astrocytes (Ast), neurons, oligodendrocyte precursor cells (OPC), newly formed oligodendrocytes (Immature oligos), myelinated oligodendrocytes, microglia, and endothelia cells (Zhang et al., 2014). FPKM stands for fragments per kilobase of transcript per million mapped reads. Endothelia is highlighted in red for clarity.

Article Snippet: Briefly, culture plates were coated with 5μg/mL solution of the extracellular domain of Notch ligand fused at the C-terminal to the Fc portion of human IgG1 (Dll1 (mouse):Fc (human) recombinant protein AdipoGen AG-40A-0148Y, Dll4 (mouse):Fc (human) recombinant protein AdipoGen AG-40A-0145, Jag1 (mouse):Fc (human) recombinant protein AdipoGen AG-40A-0157T, or Jag2 (mouse):Fc (human) recombinant protein R&D Systems 4748-JG).

Techniques: RNA Sequencing, Expressing, Activation Assay

Journal: Neurochemistry international

Article Title: Reciprocal communication between astrocytes and endothelial cells is required for astrocytic glutamate transporter 1 (GLT-1) expression

doi: 10.1016/j.neuint.2020.104787

Figure Lengend Snippet: A) Confluent bEND.3 cells were incubated with medium containing 5μg/mL of anti-Dll1, anti-Dll4, anti-Jag1, anti-Jag2, or an IgG of the same isotype for one hour prior to the introduction of astrocytes. The cultures were maintained for 7-10 days with complete media changes every 3-4 days. With every media change antibody was added at a final concentration of 5μg/mL. The γ-secretase inhibitor DAPT (10μM) was used as a positive control of Notch inhibition. GLT-1 and β-actin were detected on the same membranes. Representative blot and summary of quantification of GLT-1 normalized to β-actin are shown. This Western blot was cut to keep the layout of the figure similar to that shown in Fig 1. Data are the mean ± SEM of 6 independent experiments **** p < 0.0001 indicates comparison to astrocyte monocultures (control); $$ p < 0.01, #### p < 0.0001 for indicated comparisons. B) bEND.3 cells were infected with lentiviral vectors that contained shRNA directed against Dll1, Dll4, or a scrambled shRNA control (shScr), 2-3 days after plating. The media was replaced with fresh media 24 hours later, and the next day astrocytes were layered on top of transduced bEND.3 cells. Cells were harvested 7-10 later and GLT-1 and β-actin were detected on the same membranes. Representative blots and summary of quantification of GLT-1 normalized to β-actin are shown. Data are the mean ± SEM of 5 independent experiments. ***p < 0.001, **** p < 0.0001 indicates comparison to astrocyte monocultures, #### p < 0.0001 for indicated comparison, $ p < 0.05 indicate comparisons to shScr.

Article Snippet: Briefly, culture plates were coated with 5μg/mL solution of the extracellular domain of Notch ligand fused at the C-terminal to the Fc portion of human IgG1 (Dll1 (mouse):Fc (human) recombinant protein AdipoGen AG-40A-0148Y, Dll4 (mouse):Fc (human) recombinant protein AdipoGen AG-40A-0145, Jag1 (mouse):Fc (human) recombinant protein AdipoGen AG-40A-0157T, or Jag2 (mouse):Fc (human) recombinant protein R&D Systems 4748-JG).

Techniques: Incubation, Concentration Assay, Positive Control, Inhibition, Western Blot, Comparison, Control, Infection, shRNA