Figure 1. E-cadΔ70/α homodimerization is... | Rockefeller University Press

Figure 1.

$Figure 1. E-cadΔ70/α homodimerization is required for robust interaction with F-actin. (A) Schematic representation of the E-cadherin/αE-catenin chimeras. CBD, β-catenin-binding domain. (B) Ion exchange chromatography (IEC) of recombinant E-cadΔ70/α, and SDS-PAGE of protein from the resulting two peaks (fractions indicated in purple and green) stained with Coomassie Brilliant Blue (CBB). (C) Superdex 200 size exclusion chromatography of the two peaks from the IEC shown in B. Fractions indicated with a bracket were pooled and analyzed by Native-PAGE, and stained with CBB. (D) CBB stained Native-PAGE of increasing concentrations of monomeric E-cadΔ70/α chimera incubated for 16 h at 37°C. Ctrl, purified monomeric chimera. Quantification of the percentage of dimerization with standard deviation from three independent experiments. (E) Coimmunoprecipitation of Myc-tagged E-cadΔ70/α with HA-tagged E-cadΔ70/α from transfected L cells. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. A representative image of three independent experiments is shown. (F) High-speed co-sedimentation of F-actin with E-cadΔ70/α monomer (purple) or homodimer (green). The data shown are from a single representative experiment out of three independent experiments. (G) Pyrene–actin polymerization assay with 10% pyrene–actin (white), with Arp2/3 complex and WASp-VCA (black), and either 8 µM E-cadΔ70/α homodimer (green) or monomer (purple). The data shown are from a single representative experiment out of three independent experiments.$

E-cadΔ70/α homodimerization is required for robust interaction with F-actin. (A) Schematic representation of the E-cadherin/αE-catenin chimeras. CBD, β-catenin-binding domain. (B) Ion exchange chromatography (IEC) of recombinant E-cadΔ70/α, and SDS-PAGE of protein from the resulting two peaks (fractions indicated in purple and green) stained with Coomassie Brilliant Blue (CBB). (C) Superdex 200 size exclusion chromatography of the two peaks from the IEC shown in B. Fractions indicated with a bracket were pooled and analyzed by Native-PAGE, and stained with CBB. (D) CBB stained Native-PAGE of increasing concentrations of monomeric E-cadΔ70/α chimera incubated for 16 h at 37°C. Ctrl, purified monomeric chimera. Quantification of the percentage of dimerization with standard deviation from three independent experiments. (E) Coimmunoprecipitation of Myc-tagged E-cadΔ70/α with HA-tagged E-cadΔ70/α from transfected L cells. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted for HA and Myc. A representative image of three independent experiments is shown. (F) High-speed co-sedimentation of F-actin with E-cadΔ70/α monomer (purple) or homodimer (green). The data shown are from a single representative experiment out of three independent experiments. (G) Pyrene–actin polymerization assay with 10% pyrene–actin (white), with Arp2/3 complex and WASp-VCA (black), and either 8 µM E-cadΔ70/α homodimer (green) or monomer (purple). The data shown are from a single representative experiment out of three independent experiments.

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