Interaction of Human Notch1 with Human DLL4.
HEK293 cells transfected with a Human Notch1 or a Human GITR ligand expressing vector were incubated with 25 μg/ml of Human GITR-Fc or ab108557. Cells were stained with anti-Human IgG (Fc specific) FITC conjugate for DLL4-Fc binding.

Induction of Hes-1 with the treatment of recombinant Human DLL4-Fc (ab108557).

A Mouse preadpipocyte cell line, 3T3L-1, was stimulated with 5 μg/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.

M: Marker.
Lane 1: hDLL4-Fc, 0 min.
Lane 2: hDLL4-Fc, 10 min.
Lane 3: hDLL4-Fc, 30 min.
Lane 4: hDLL4-Fc, 1 hr.
Lane 5: hDLL4-Fc, 2 hr.
Lane 6: hDLL4-Fc, 4 hr.
Lane 7: hDLL4-Fc, 8 hr.
Lane 8: hDLL4-Fc, 24 hr.

Adipogenesis inhibition of 3T3L-1 cells.
3T3L-1 cells (mouse pre-adipocyte cells) were maintained in DMEM, supplemented with 10% fetal bovine serum and penicillin-streptomycin. For differentiation of 3T3L-1 cells, 3T3L-1 cells were cultured in adipogenic medium which was growth medium supplemented with 1 μM Dexamethasone, 0.5 mM IBMX, 10 μg/ml lnsulin (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 (20 ng/ml) was added. Recombinant Human DLL4-Fc (ab108557) (5 μg/ml) dissolved in DPBS was added to the differentiation medium. These plates were then used to differentiate 3T3L-1 cells.

Adipogenesis inhibition of MSCs.
MSCs (Mesenchymal stem cells) were maintained in DMEM, supplemented with 10% fetal bovine serum, penicilinstreptomycin and glutamine. For differentiation of MSCs, MSCs were cultured in adipogenic medium which was growth medium supplemented with 1 μM Dexamethasone, 0.5mM IBMX, 10 μg/m lnsulin, 100 μM 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 (20 ng/ml) was added. To immobilize Notch ligands on the plastic surface of the culture plates, plates were incubated with a solution of ab108557 (5 μg/ml) or mCD137-Fc (5 μg/ml) in PBS for 2 hours at 37°C. Plates were then used to differentiate MSCs.

Adipogenesis inhibition of 3T3L-1 cells.

50 μg of cell lysates derived from hDLL4-Fc (ab108557) or non-treated 3T3L-1 cells, which had been either differentiated or undifferentiated, and were subjected to Western blot by using a Mouse adiponectin antibody.