Figure 3
From: Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity
VEGFSWITCH-ON bioactivity.
(a) Comparison of VEGFR2 phosphorylation induced by VEGFSWITCH-ON, VEGF and VEGFGyrB with GyrB as a negative control. HUVECs were stimulated for 15 min with the indicated protein concentrations after which whole-cell extracts were subjected to SDS-PAGE followed by Western blot analysis using an antibody against phosphorylated VEGFR2 (P-VEGFR2 (Tyr 1175))18. Subsequently, the same membrane was re-probed with an anti-VEGFR2 antibody18 for detection of the total levels of VEGFR2. (b) Stimulation of DLL4 expression. HUVECs were stimulated for 4 days with the indicated VEGFSWITCH-ON concentrations after which whole-cell extracts were resolved by SDS-PAGE followed by Western blot analysis. After transfer of the proteins to a PVDF-membrane, the membrane was cut into two pieces. The upper part of the membrane (containing proteins with a molecular weight larger than 60 kDa) was probed with an anti-DLL4 antibody19. As a loading control, the levels of β-actin on the lower part of the membrane (containing proteins with a molecular weight below 60 kDa) were detected using an anti-β-actin antibody20.
