CREB negatively regulates RhoA signaling. (A) Effects of CREB depletion on RhoA (top row and panel B) or myosin phosphatase 1 (MYPT1; second row and panel C) activities. HPAE cells transfected with either SiSc or SiCREB were left unstimulated or stimulated with 50nM thrombin for indicated times after which they were lysed to measure RhoA activity. MYPT1 activity was determined by immunoblotting with anti–phospho-381 MYPT antibody. Top row shows RhoA activity as indicated by the amount of RBD-bound RhoA, whereas third row shows total amount of RhoA protein in whole cell lysate. Bottom row shows immunoblot of CREB using anti-CREB antibodies. (B-C) Plots show mean + SD of fold-increase in RhoA (B) or MYPT1 (C) activities from multiple experiments over value at time 0 (n = 4). Asterisk (*) indicates values different from unstimulated SiSc group (P < .05), and double asterisk (**) indicates values different from corresponding thrombin-stimulated, SiSc-transfected monolayers (P < .05).