Fig. 8.

GSK-3 β mediates MLC activation in inducible Rcan1^−/− mice. a Experimental design. Protein extracts from brains of Rcan1^+/+ and Rcan1^−/− mice were immunoprecipitated with anti-Rcan1 antibodies, and interacting proteins were identified by mass spectrometry and quantified by spectral counting. b Significantly enriched proteins (Student t-test, p < 0.05) identified in Rcan1 immunoprecipitates from Rcan1^+/+ and Rcan1^−/− extracts are listed according to their enrichment in 3 independent experiments. c Gsk-3 β and Rcan1 immunoblot analysis of vSMC protein extracts immunoprecipitated with anti-Rcan1 antibody (IP Rcan1) or control antibody (IP IgG), compared with crude extract (Input). d Representative p-MLC, β-catenin, and tubulin (loading control) immunoblot analysis of aortic extracts from Rcan1^+/+, SM-Rcan1^−/−, and EC-Rcan1^−/− mice (n = 3 independent experiments). e Representative β-catenin, and tubulin (loading control) immunoblot analysis of aortic extracts from Rcan1^+/+ (n = 7), Rcan1^−/− (n = 6) and SM-Rcan1^−/− (n = 8) mice. d, e Rcan1^+/+ littermates consisted of a pool of vehicle-treated Cre-positive and tamoxifen-treated Cre-negative Rcan1^fl/fl mice. f Representative images (n = 3) of p-MLC immunofluorescence (gray) and DAPI staining (blue) in Rcan1^fl/fl vSMCs transduced with Cre-encoding or control lentivirus and treated for 2 h with saline or the GSK-3 β inhibitors LiCl (50 µM) or inhibitor VIII (GSK-3 βi) (10 µM). Scale bar, 20 µm. g Representative Rcan1, p-MLC, and β-catenin immunoblot analysis (n = 3) in vSMCs from f. Gapdh was used as loading control. h Model depicting the GSK-3 β- and ROCK-mediated induction of aortic IMH, aortic dissection, and AAA in conditional Rcan1^−/− mice subjected to hypertensive stress. While hypertension induces spontaneously-regressing small IMH in Rcan1^+/+ mice at low frequency, in most conditional Rcan1^−/− mice it induces large IMH that dissect or progress to aneurysm