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

Fig. 4.

Fig. 4.

Rescue of EGKO ECs with Gab1, eNOS, and PKA restores VEGF signaling and tube formation. The ECs from the control and EGKO mice, or EGKO ECs overexpressing vector, Gab1, constitutively active eNOS (eNOS S1176D) or constitutively active PKA (caPKA) were serum-starved and stimulated with VEGF (50 ng/mL) for indicated time periods, followed by immunoblot analysis (A and B), NO release measurement (C), and Matrigel tube formation assay (D). (A) Immunoblots of phosphorylations of eNOS and PKA substrate, and of Gab1 and PKA Cα, in control cells, parental, and vector-, Gab1- or caPKA-rescued EGKO cells after VEGF stimulation. The differences in P-eNOS levels of Gab1- or caPKA-rescued EGKO cells were significant compared with vector rescued EGKO cells (P < 0.01). (B) Immunoblot of eNOS in parental EGKO cells or vector- or eNOS S1176D-introduced EGKO cells. (C) Results of NO release measurement of parental EGKO cells, or EGKO cells introduced with Gab1, eNOS S1176D, constitutively active PKA, or empty vector, pretreated with or without PKI (10 μM) before VEGF stimulation. (E) Matrigel tube formation analysis of parental EGKO cells, or EGKO cells introduced with Gab1, eNOS S1176D, constitutively active PKA, or empty vector, pretreated with or without PKI (10 μM) before VEGF stimulation. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01) for EGKO versus control.