Skip to main content
. 2011 Jan 31;108(7):2957–2962. doi: 10.1073/pnas.1009395108

Fig. 3.

Fig. 3.

Gab1-deficient ECs are defective in VEGF signaling and tube formation. ECs isolated from EGKO and control mice were serum-starved and stimulated with VEGF (50 ng/mL) for indicated time periods, followed by analyses of in vitro endothelial Matrigel (A) and wound healing (B) assays, immunoblot (C and D), and NO release measurement (E). (A) Representative results of Matrigel tube formation assay. Arrows indicate endothelial tube branches. (B) Results of endothelial wound healing assay (*P < 0.05 vs. control). (C) Immunoblots of phosphorylations of eNOS, Akt, Erk1/2, and AMPK in EGKO and control ECs. The relative band intensities, which represent the average of three independent experiments (the same is true for all of the gel quantification data in the following figures), of phosphorylated eNOS (P-eNOS) and phosphorylated Akt (P-Akt) are indicated under each band. The differences in the levels of P-eNOS and P-Akt of EGKO and control groups were significant (P < 0.02). (D) Immunoblots of phosphorylations of eNOS and PKA substrate, and of PKA Cα and Cβ subunits in EGKO and control ECs, pretreated with or without myristoylated PKI 14–22 amide (PKI; 10 μM), a specific PKA inhibitor, before VEGF stimulation. Arrows indicate phosphorylation bands of PKA substrates, which are PKI-sensitive, and substantial decrease in EGKO ECs compared with control cells. The difference in P-eNOS levels of EGKO and control groups was significant (P < 0.01). (E) NO release measurement of EGKO ECs pretreated with or without PKI (10 μM) before VEGF stimulation (**P < 0.01 vs. control.) (F) Representative results of Matrigel tube formation assay of EGKO and control ECs, pretreated with or without N-nitro-L-arginine methyl ester (L-NAME; 5 mM), an inhibitor of eNOS, and PKI (10 μM) before VEGF stimulation (50 ng/mL). (G) Immunoblots of phosphorylation of eNOS, Akt, Erk1/2, AMPK, and PKA substrate in EGKO and control aortic rings after VEGF stimulation (100 ng/mL). The differences in the levels of P-eNOS and P-Akt of EGKO and control groups were significant (P < 0.02). Asterisks indicate statistical significance (*P < 0.05, **P < 0.01) for EGKO versus control.