Figure 4: GRAF3 depletion in cultured SMC promotes Rho activity.
From: The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertension
(a) Ectopic expression of myc-GRAF3 (arrow, left; blue, right) in rat aortic SMC (RAoSMC) induced marked clearing of actin stress fibres as assessed by phalloidin staining (white, left or green, right) and focal adhesions as marked by vinculin (white, left or red, right). (b) Levels of GRAF3 and GAPDH mRNA in control (Csi) and GRAF3 (G3) si-treated RAoSMC. Data are representative of seven separate experiments. (c) GRAF3 si-treated RAoSMC exhibited a more contractile morphology (more abundant vinculin-labeled focal adhesions and phalloidin-labeled stress fibres) and elevated phospho-MLC (pMLC) when compared with control si (Csi)-treated cells. Scale bars for panels a and c are 50 microns. (d) GRAF3 si and Csi-treated RAoSMC were stimulated with S1P and active (GTP-bound) RhoA was precipitated with GST-rhotekin beads and assessed by immunoblotting (top panel). Densitometric analysis of three separate experiments is presented as mean±s.e.m. (bottom panel). P values (Student’s t-test) are expressed as: †P<0.1; *P<0.05.
