JCS019646 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Pull-down screening for molecules interacting with the ASD2 domain of Shroom3. GST-tagged Shrm3-ASD2 or GST was incubated with E9.5 mouse embryonic lysates and pulled down with Glutathione-Sepharose 4B beads. Pulled-down proteins were visualized by silver staining, and subsequently analyzed by mass spectrometry. Asterisks show the protein bands of GST-tagged molecules. Mass spectrometry analysis was performed at the Mass Spectrometry Analysis Laboratory of our institute.

• Supplemental Figure S2 -

  Fig. S2. R1-C1 fragment of Rock1 interacts with ASD2, and can antagonize the endogenous ROCK-Shroom3 interaction. (A) Top, schematic representation of Rock1 and its deletion mutants. Bottom, interaction of ASD2 with Rock1 deletion mutants. COS7 cells were co-transfected with FLAG-tagged ASD2 and HA-tagged Rock1 (RI-Full) or its deletion mutants. Cell lysates were prepared and then subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA or anti-FLAG antibody. Only the constructs containing the region from a.a. 726 to 926 bind ASD2. (B) Expression of RI-C1 interferes with the action of Shroom3. EGFP-tagged RI-C1 was expressed in MDCK-Tet-Off Shroom3 transfectants. The apical junctions are visualized with anti-ZO1 antibody in red. RI-C1 (green) is localized at the junctions only when Shroom3 expression has been induced, indicated by an arrow. 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; bars represent the standard deviation. *P<0.01 against the EGFP-transfected cells. Scale bars: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Schematic representation of electroporation of RNAi plasmids into chicken embryos.

• Supplemental Figure S4 -

  Fig. S4. Cell assembly at the apical surface of developing neural plate and tube. (A) Neural tubes of stage 9 chicken embryos were dissected, fixed and double-immunostained for ZO-1 and myosin 2A or Par-3. These proteins co-localize together, in contrast with the case of pMLC. (B) The dorsal ectoderm at the future head region in a stage 7 embryo was double-immunostained for ZO-1 and pMLC. Some rosette-like cell clusters are encircled. Scale bars: 10 µm.

• Supplemental Figure S5 -

  Fig. S5. Characterization of antibodies raised against Shroom3 and Rock1. (A) Rabbit antibody raised against mouse Shroom3. Lysates of MDCK-Tet-Off cells inducibly expressing FLAG-tagged Shroom3 were immunoblotted with the anti-Shroom3, anti-FLAG or anti-α-tubulin antibody. (B) Rat antibody raised against mouse Rock1. Left, lysates of COS7 cells expressing HA-tagged Rock1 or Rock2, or vector alone were subjected to immunoprecipitation with anti-HA antibody, and then immunoblotted with rat anti-Rock1 or anti-HA antibodies. Right, lysates of MDCK cells and COS7 cells were immunoblotted with the rat anti-Rock1 antibody. Immunoblot detection of HA-tagged Rock1 with anti-HA antibody is also shown. (C) Rat antibody raised against chicken Rock1. GST-tagged chicken Rock1 and Rock2 antigens (left), and a chicken embryonic lysate (right) were immunoblotted with the rat anti-chicken Rock1 antibody.