Figure 5.

NZO-3 does not interact with RhoA, Rac, or Cdc42 from MDCK cell lysates; CZO-3 binds AF-6 and p120 catenin. (A) MDCK cell lysate was added to affinity columns containing immobilized GST-NZO-3 or GST alone, and the bound fractions were immunoblotted with anti-RhoA, anti-Rac, and anti-Cdc42 antibodies. Presence of each GTPase in the lysate was confirmed by immunoblotting an aliquot of lysate. None of the GTPases tested were retained by NZO-3 or GST alone. (B) Equivalent amounts of full-length ZO-3 (FLZO-3), NZO-3, CZO-3 or GST alone immobilized on glutathione-Sepharose were incubated with MDCK lysate. The bound fraction was immunoblotted with anti-p120 catenin or anti-AF-6 antibodies. Both p120 catenin and AF-6 bind to FLZO-3 and CZO-3, with negligible binding to NZO-3. (C) Equivalent amounts of GST or GST-p120 were immobilized on glutathione-Sepharose beads; purified 6-histidine–tagged CZO-3 was then added to each affinity column. The bound fractions were immunoblotted with an antibody that recognizes the C terminus of ZO-3. CZO-3 is specifically retained on GST-p120 beads, and not to GST alone.