Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells
Fig 1
(A) Co-immunoprecipitation assays. YFP-Solo was expressed in MCF10A cells and the cell lysates were immunoprecipitated (IP) with an anti-GFP antibody and analyzed by immunoblotting with anti-GFP and anti-β4 antibodies. (B–E) Mapping of the binding regions of Solo and β4. (B) Schematic domain structure of β4 and its deletion mutants used in this study. Numbers denote amino acid residues flanking each region. The binding ability of each fragment to FLAG-Solo is indicated in the right column. Conserved domains are denoted as: vWFA, von Willebrand factor type A; EGF, EGF-like; Calx, Calx-beta; FNIII, fibronectin type III; CS, connecting segment. (C) Co-immunoprecipitation assays of β4 fragments with Solo. YFP-tagged β4 fragment (β4-YFP) and FLAG-tagged Solo-WT were co-expressed in COS-7 cells, and the cell lysates were immunoprecipitated with an anti-FLAG antibody and analyzed by immunoblotting with anti-FLAG and anti-GFP antibodies. Arrowheads indicate the expected positions of YFP-tagged β4 fragments. (D) Schematic domain structure of Solo and its deletion mutants used in this study. The binding ability of each fragment to β4 (1451–1752)-YFP is indicated in the right column. Conserved domains are indicated as Solo, CRAL/TRIO, SPEC (spectrin repeats), DH, and PH domains. (E) Co-immunoprecipitation assays of Solo fragments with β4. FLAG-Solo or its fragments were co-expressed with β4 (1451–1752)-YFP in COS-7 cells, and the cell lysates were immunoprecipitated with an anti-FLAG antibody and analyzed by immunoblotting with anti-FLAG and anti-GFP antibodies. (A, C, and E) These experiments were repeated more than three times and reproducible results were obtained.