Physical interaction of CCM2 and KRIT1 is required for their endothelial junctional localization and for inhibition of RhoA→ROCK-mediated permeability. HUVECs were transfected with siRNA to deplete endogenous CCM2 and reconstituted with either WT CCM2 or CCM2-F217A, a mutation which abrogates CCM2 binding to KRIT1. Both WT CCM2 and CCM2-F217A constructs bear silent mutations conferring resistance to CCM2 siRNA. (A) Endogenous CCM2 and KRIT1 protein are localized to HUVEC endothelial cell–cell junctions in cells treated with control siRNA. CCM2 depletion by siRNA causes KRIT1 loss from junctions. Reconstitution of depleted endogenous CCM2 by transfection with CCM2-F217A does not restore junctional localization either to the mutant CCM2 or to endogenous KRIT1. Reexpressed WT CCM2 is junctional and permits endogenous KRIT1 junctional localization. (B) CCM2-F217A reconstitution of endogenous CCM2 depletion fails to suppress RhoA activity, whereas reconstitution with WT CCM2 limits RhoA activity. Error bars are means ± SE (n = 4). *, P < 0.01 compared with vector-treated control siRNA. (C) Transfection and knockdown efficacy for A and B is shown by Western blotting for CCM2 and total RhoA content. Equal loading is shown by staining for GAPDH. 1, vector; 2, vector + CCM2 siRNA; 3, CCM2 cDNA; 4, CCM2 cDNA⁺ CCM2 siRNA; 5, CCM2-F217A cDNA; 6, CCM2-F217A cDNA + CCM2 siRNA; 7, control rabbit IgG IP. (D) CCM2 depletion causes increased pMLC and actin stress fiber content, reversible by reconstitution with WT CCM2 but not by CCM2-F217A, indicating a requirement for CCM2-KRIT1 physical interaction for suppressing ROCK activity. Bar, 50 µm. (E) CCM2 depletion-induced hyperpermeability is not reversible by CCM2-F217A reconstitution. Error bars are means ± SE (n = 6). *, P < 0.001 compared with control siRNA.