p38MAPK induction by VEGF-A165 and VEGF-E-NZ2, but not by VEGF-A121. (A) PAE/VEGFR-2, NRP1 cells were incubated for different time periods with 2 nM of VEGF-A165, VEGF-A121, or VEGF-E-NZ2, followed by analyses, as indicated, for induction of p38MAPK, phospho (p)Erk, pAkt, pPLCγ, and pVEGFR-2 as well as unphosphorylated counterparts. An exogenous substrate (ATF-2⁵⁰ ) was used to measure p38MAPK activity, whereas phospho-specific antibodies were used to measure induction of Erk, Akt, PLCγ, and VEGFR-2. (B) Quantification of immunoblots shown in panel A, of induced bands at the time point of maximal induction (3, 5, or 10 minutes; see blot in panel A), in relation to relevant controls (eg, p38MAPK activity in relation to p38MAPK loading control, etc). The effect of VEGF-A165 is set to 1 for the different conditions. (C) 3D embryoid body cultures treated with VEGF-A165 in the absence and presence of 5 or 10 μM SB203580 show dose-dependent reduction in angiogenic sprouting (arrows). Scale bar represents 300 μm. (D) Quantification of CD31-sprout area in panel C. (E) Endothelial cell organization and pericyte association in subcutaneous matrigel plugs containing VEGF-A165 (left panels) or VEGF-A165 combined with 10 μM of SB203580 (right panels). Plugs were retrieved after 7 days and analyzed by whole-mount immunostaining to detect CD31 (red) and ASMA (green) followed by confocal microscopy. Microphotographs show projections of z-stacks of 90 μm. Scale bar represents 50 μm. (F) Quantification by CD31-positive area in VEGF-A165 and VEGF-A165/10 μM of SB203580-containing matrigel plugs.