Fig. 9. Shroom2 binds to ZO-1 in vitro and in vivo. (A) Domain structure of the shroom2 yeast two-hybrid bait (mShrm2PSP) and of the two ZO-1 preys isolated. (B) Several ³⁵S-labeled shroom2 fragments (labeled 1 to 6) were incubated with the GST-tagged ZO-1 prey (GST-ZO1 prey) or GST alone. mShrm2, mShrm2PDZ, mShrm2PSP and mShrm2SPR bind to the GST-ZO1 prey, but not to GST. By contrast, mShrm2ASD2 and mShrm2PDZ do not bind to the GST-ZO1 prey. (C) Characterization of the shroom2-interaction domain of ZO-1. ³⁵S-labeled shroom2 and ³⁵S-labeled shroom2-SPR only bind to the original GST-ZO1 prey and to the GST-ZO1 SH3/GuK fragment. (D) Protein extracts from the mouse auditory organ (left panels) or brain (right panels) were used for the immunoprecipitation assays. The endogenous shroom2 protein is immunoprecipitated in the presence of the anti-ZO-1 antibody, but not with protein G alone. Under the conditions we used, ZO-1 immunoprecipitates do not contain myosin VIIa (bottom panels).