FIG. 2.

The Abi1 HHR domain is required for binding to Nap1 and formation of cell-cell junctions. (A) Untransfected A431 cells or A431 cells expressing GFP-Nap1 were stained with the indicated antibodies, and confocal images were merged in the right panels. Abi1, Nap1, and β-catenin show complete colocalization at cell-cell junctions. (B) EYFP-Abi1 or the indicated EYFP-Abi1 mutants were cotransfected into 293T cells with Flag-Nap1. Nap1 protein was immunoprecipitated (IP) with anti-Flag antibodies. Immunoprecipitates were analyzed by immunoblotting (IB) with anti-GFP antibodies to detect coprecipitating Abi1 proteins (top panel) and for Flag-Nap1 with anti-Flag antibodies (middle panel). Total lysates were analyzed for expression of EGFP-Abi1 proteins by blotting with anti-GFP antibodies (bottom panel). The locations of the IgG heavy chain and the Abi1 Δ277-446 protein (*) are indicated. (C) Schematic diagram of Abi1. The core of the Nap1-binding domain is underlined and corresponds to the HHR domain. (D) A431 cells were transduced with retroviruses coding for GFP, GFP-Abi1 WT, or GFP-Abi1 ΔHHR as indicated. Cells were sorted for GFP by FACS, and cells expressing high GFP levels were fixed and stained with anti-β-catenin antibody (bar, 10 μm). (E) The percentages of cells with cell-cell junctions of randomly selected regions were analyzed. Results represent the means ± SEMs of at least three independent experiments, and at least 500 cells were counted in each experiment. *, P < 0.01.