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Figure 1


Fig. 1. Shroom3 binds ROCKs via its ASD2 domain. (A) Schematic representation of Shroom3 and its deletion mutants. (B) MDCK-Tet-Off cell lines inducibly expressing FLAG-tagged, full-length Shroom3 (Shrm-Full) or Shroom3-{Delta}ASD2 (Shrm-{Delta}ASD2) were double-immunostained for FLAG and ZO-1. Expression of these molecules is suppressed in the presence of doxycycline [Dox(+)], but induced by its removal (Dox[-]) from the culture medium. The apical cell junctions, delineated by ZO-1 signals, are tightened by expression of Shrm-Full but not of Shrm-{Delta}ASD2. Scale bars: 10 µm. (C) In vitro interaction of ASD2 with endogenous Rock1 or Rock2. GST-tagged Shrm-ASD2 or GST was incubated with a MDCK cell lysate, and then pulled down with Glutathione-Sepharose 4B beads, followed by immunoblotting with antibodies against Rock1, Rock2, {alpha}-tubulin and GST. (D) In situ interaction of ASD2 with Rock1 or Rock2. COS7 cells were co-transfected with FLAG-tagged Shrm-ASD2 and HA-tagged Rock1 or Rock2. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA or anti-FLAG antibody.





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