Fig. 3. A central fragment of ROCK interacts with ASD2, and can antagonize the endogenous ROCK-Shroom3 interaction. (A) (Top) Schematic representation of Rock2 and its deletion mutants. (Bottom) Interaction of ASD2 with Rock2 deletion mutants. COS7 cells were co-transfected with FLAG-tagged ASD2 and HA-tagged Rock2 (RII-Full) or its deletion mutants. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA or anti-FLAG antibody. Only the constructs containing the region from amino acids 698 to 957 can bind ASD2. See Fig. S2 in the supplementary material for the corresponding experiments for Rock1. (B) Expression of the central region of Rock2 (RII-C1) interferes with the action of Shroom3. EGFP-tagged RII-C1 or RII-C2 was expressed in MDCK-Tet-Off Shroom3 transfectants. The apical junctions were visualized with anti-ZO1 antibody (red). RII-C1 (green) is localized at the junctions only when Shroom3 expression has been induced by Dox(-), as indicated by arrows. Bar graphs show quantitative measurement of the length of apical junctions and apical surface areas in the same cultures. A total of 100-200 of GFP-positive cells were counted. Histograms are the average of three independent experiments, and bars represent the standard deviation. ^*P<0.01 against the EGFP-transfected cells. P-values were analyzed by Student's t-test throughout the manuscript. (C) RII-C1 interferes with the ROCK-Shroom3 interaction. (Left) MDCK-Tet-Off FLAG-Shrm-Full transfectants were further transfected with either EGFP-tagged RII-C1 or EGFP. From their lysates, Shroom3 was immunoprecipitated with anti-FLAG antibody, which was followed by detection of endogenous Rock1 or EGFP. Black triangle, EGFP-RII-C1; white triangle, EGFP. (Right) Localization of endogenous Rock1 or pMLC in Shroom3-expressing cells, in which restricted cells had been transfected with EGFP-RII-C1. Junctional localization of those molecules is inhibited in the RII-C1-positive cells. Scale bars: 10 µm.