Fig. 5

Nova2 Regulates MAPK-ERK Signaling

(A and B) Phospho-Erk1/2 (pErk) immunostaining at 28 hpf (A) and 32 hpf (B) in Tg(fli1a:nEGFP)^y7 siblings and nova2^uq14bh mutants. Arrowheads indicate pErk-positive ECs (brackets indicate PCV).

(C) Quantification of the number of pErk-positive ECs in the dorsal side of the PCV across 7 body segments at 28 hpf (siblings n = 20 and mutants n = 12) and 32 hpf (siblings n = 29 and mutants n = 20).

(D) Quantification of the number of pErk-positive neurons across 3 body segments at 28 hpf. (siblings n = 20 and mutants n = 12).

(E) Representative immunostains (32 hpf) for pSrc (upper, no qualitative differences observed) and for pSmad1/5/8 (lower, in which pSmad-positive arterial ECs and venous ECs were scored in the DA and PCV). No difference in the number of pSmad-positive cells across 4 somites was seen in DA (sib: 3.6 ± 1.6 [SD, n = 9], mut:4 ± 1.3 [n = 10]) or PCV (sib: 1.8 ± 1.5 [n = 9], mut:1.4 ± 1.3 [n = 10]).

(F) Western blot analysis of ERK activation upon NOVA2knockdown in HuLECs stimulated with VEGFC. Representative of four independent blots, lower bands non-specific in both blots.

(G) Quantitative analysis of normalized pERK expression (pErk/total Erk) relative to control siRNA levels from (F) shows prolonged activation of ERK at 30 min following VEGFC treatment.

(H) Summary of phosphoarray analysis from human LECs. Left: Summary of overall experimental outcome with n = 41 unchanged phospho-sites, n = 31 increased phosphorylation events and n = 13 dephosphorylationevents. Right: Representative heat map of phosphoarray outcome. Increased and decreased phosphorylation events and targets are indicated. Data generated from n = 2 independent arrays with 6 independent assays for each Ab on each array.

Error bars: SD, Scale bars: 25 μm, t test (D and G) or Mann-Whitney-test (C), ^∗p < 0.05, ^∗∗p < 0.01, and ns, not significant.

See also Figure S5.