Extended Data Figure 2. Endothelial Rho activity, but not β-catenin or SMAD3 signaling, is increased during CCM formation.

a, Immunostaining for the endothelial marker PECAM and pMLC in the white matter (“WM”) of the cerebellum of P6 control and Krit1^ECKO littermates is shown. Scale bars, 50 μm. b, Anti-GFP immunostaining to detect TCF/Lef:H2B-GFP Wnt/β-catenin reporter (“WntREP”) activity. Scale bars, 50 μm. c, Immunoblotting of P6 brain endothelial cell lysate for GFP. Results are representative of 3 separate experiments. d, qPCR analysis of β-catenin target genes in hindbrain endothelial cells. N=4–5; P>0.05 for comparison of all values. Error bars indicate SEM. e, Immunostaining for phospho-SMAD3 (“pSMAD3”) and PECAM. Scale bars, 50 μm. f, Total SMAD3 and pSMAD3 were detected by immunoblotting cerebellar endothelial cell lysate. Results are representative of 3 separate experiments.