Dll4 is incorporated into exosomes. (A) HUVECs were seeded onto 6-well plates at 3 × 105/well with or without the presence of 10nM dibenzazepine (DBZ). The plates had been coated previously with either bovine serum albumin (BSA; 1 μg/mL) or recombinant hDll4 (1 μg/mL) to induce Notch signaling. The cells were lysed after 24 hours and subjected to Western blotting to detect Dll4 or β-actin as a loading control. (B) HUVECs were grown on recombinant hDll4–coated plates for 48 hours in exosome-depleted EGM2. The medium was harvested, and the cells were lysed in radioimmunoprecipitation assay buffer. The medium was ultracentrifuged to isolate the exosomes (exo), then the fractions were analyzed by Western blotting to detect Dll4. (C) U87 Dll4 and U87 control cells were grown for 48 hours in Opti-Mem before harvesting of the media and cell lysis. The exosomes were harvested by ultracentrifugation and subjected to Western blotting to detect Dll4 and exosomal markers. (D) Exosome size was analyzed with a NanoSight LM10; vertical axis units 1 × 107 particles/mL per nanometer.
