DLL4 (human):Fc (human), (recombinant)

Notch ligand



ALX-201-386-C010	10 µg	292.00 USD

ALX-201-386-C050	50 µg	826.00 USD

Product Specification

Alternative Name:	Delta-like protein 4, Delta4

Concentration:	~0.25mg/ml

Endotoxin Content:	<0.1EU/µg protein (LAL test).

Purity:	≥90% (SDS-PAGE)

Formulation:	Liquid. 0.2µm-filtered solution in PBS.

Activity:	Interacts with human Notch1 (as confirmed by flow cytometry).

Source/Host:	Produced in HEK 293 cells. Extracellular domain of recombinant human DLL4 (Delta-like protein 4; Delta4) (aa 1-529) is fused at the C-terminus to the Fc portion of human IgG.

Long Term Storage:	-20°C

Use/Stability:	Working aliquots are stable for up to 3 months when stored at -20°C.

Handling:	After opening, prepare aliquots and store at -20°C. Avoid freeze/thaw cycles.

Miscellaneous/General:	Notch signalling pathways play key roles in cell-fate determination and differentiation in many tissues during embryonic and postnatal development. Notch ligands are divided into two subclasses, the delta and the serrate family. DLL4 (Dll4) is a delta family member and exhibits all the hallmarks of the delta family of Notch ligands. Human DLL4 shares 86% aa sequence identity with mouse Dll4. The extracellular region of the 685 aa protein contains 8 EGF-like repeats as well as a DSL (Delta, Serrate, Lag2) domain necessary for receptor binding. The protein also contains a transmembrane domain and a cytoplasmic tail lacking any catalytic motifs. DLL4 is largely restricted to the vascular endothelium and has been shown to function as a ligand for Notch1 and Notch4. Signalling through Notch1 regulates formation of tip cells during angiogenesis.

Background / Technical Information:	UniProt ID Q9NR61: DLL4 (human)

Figure 1: SDS-PAGE of DLL4 (human):Fc (human) (rec.) (Prod. No. ALX-201-386).

Figure 2: Differentiation of human mesenchymal stem cells (MSCs) into adipocytes in the presence or absence of Notch ligands that inhibit adipogenic differentiation of MSCs.
Method: MSCs were maintained in DMEM, supplemented with 10% fetal bovine serum, penicilin-streptomycin and glutamine. For differentiation of MSCs they were cultured in adipogenic medium corresponding to growth medium supplemented with 1μM dexamethasone, 0.5mM IBMX. 10μg/m insulin, 100μM indomethacin (day 1). Medium was changed every 3 days. Staining with Oil Red O was typically performed on day 30. For negative controls, TNF-α (20ng/ml) was added. To immobilize Notch ligands on the plastic surface of the culture plates, plates were incubated with a solution of DLL4 (human):Fc (human) (rec.) (5µg/ml) (Prod. No. ALX-201-386) or mouse CD137L:Fc (5µg/ml) in PBS for 2 hours at 37°C. These plates were then used to differentiate MSCs.

Figure 3: Adipogenesis inhibition in 3T3L-1 cells by DLL4 (human):Fc (human) (rec.) (Prod. No. ALX-201-386).
Methods:
A. 3T3L-1 cells (mouse pre-adipocyte cells) were maintained in DMEM, supplemented with 10% fetal bovine serum and penicillin-streptomycin. For differentiation, 3T3L-1 Cells were cultured in adipogenic medium which was growth medium supplemented with 1μM dexamethasone, 0.5mM IBMX, 10μg/ml insulin (day 0). Medium was changed every 2 days. Staining with Oil Red O was typically performed on day 7. Cells were washed twice with PBS, fixed with 3.7% formalin, and stained with 0.5% filtered Oil Red O in propylene glycol. For negative controls, mouse TNF-α (20ng/ml) was added. Recombinant human DLL4-Fc (5ug/ml) dissolved in DPBS was added to the differentiation medium. These plates were then used to differentiate 3T3L-1 cells.
B. 50ug of cell lysates derived from hDLL4-Fc- or non-treated 3T3L1 cells, which had been either differentiated or undifferentiated, was subjected to Western blot by means of anti-mouse adiponectin.

Figure 4: Flow cytometry confirms interaction of human Notch1 with human DLL4 (Prod. No. ALX-201-386). Method: HEK293 cells transfected with a human Notch1 or a control vector were incubated with 25μg/ml of GITR (human):Fc (human) (rec.) (Prod. No. ALX-522-061) or DLL4 (human):Fc (human) (rec.) (Prod. No. ALX-201-386). Cells were stained with anti-human IgG (Fc specific) FITC conjugate for DLL4-Fc binding.

Figure 5: Induction of Hes-1 with treatment of DLL4 (human):Fc (human) (rec.) (Prod. No. ALX-201-386).
Method: A mouse preadipocyte cell line, 3T3L1, was stimulated with 5µg/ml of human DLL4:Fc as in indicated time points and each cell lysate was prepared and subjected to Western blot by using anti-mouse Hes1 or GAPDH.

Please mouse over

General Literature References

Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting: I.B. Lobov, et al.; PNAS 104, 3219 (2007), Abstract;

Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis: M. Hellstrom, et al.; Nature 445, 776 (2007), Abstract;

Dll4, a novel Notch ligand expressed in arterial endothelium: J.R. Shutter, et al.; Genes Dev. 14, 1313 (2000), Abstract;

Human ligands of the Notch receptor: G.E. Gray, et al.; Am. J. Pathol. 154, 785 (1999), Abstract;