Figure 2. Validation of Syn^SMKO and Syn^ECKO mouse lines.

(A–C) In vivo ablation of synectin expression in smooth muscle cells (Syn^SMKO). (A) Decreased synectin RNA levels in carotid (CA) and aorta (Ao) from Syn^SMKO mice compared with control littermates. (B) Western blot of total cell lysates of aortic smooth muscle (SMC) and heart endothelial cells (EC) from Syn^SMKO mice. Note a reduction in synectin protein levels in SMC but not in EC. (C) ERK activation: Western blot analysis of total cell lysate of aortic SMC. Confluent, serum starved cells were stimulated for 5 min with 50ng/ml PDGF-BB. Phosphorylation of ERK (P-ERK) in response to PDGF-BB is not altered in synectin null smooth muscle cells (Syn^SMKO) relative to wild type smooth muscle cells (Syn^WT). T-ERK: total ERK. SM-α-actin: SMC α-actin. (D, E) In vivo ablation of synectin expression in endothelial cells (Syn^ECKO).(D) Decreased synectin protein levels in endothelial cells isolated from Syn^ECKO mice compared with Syn^WT mice. (E) Western blotting of total cell lysates isolated from heart endothelial cells (EC). Confluent, serum starved EC were stimulated for the times indicated with 50 ng/ml VEGF-A₁₆₅. Note reduced phosphorylation of ERK (P-ERK) and in VEGFR2 Y1175 site in synectin null endothelial cells (Syn^ECKO).