Purification and identification of junction-associated actin-regulatory factor complexes using a target bait-based, cross-linking approach. (a) Negative staining of MDCK membranes showing 6 nm immunogold labeling for α-actinin-4 (blue arrowheads) at sites of actin incorporation (red circle) and filament association (yellow arrowheads). (b) Immunofluorescence staining of TX-100 extracted MDCK cells for β-catenin, α-actinin-4, and actin (phalloidin) showing colocalization at junctional puncta (white arrowheads). A subset of β-catenin staining has no α-actinin-4 or actin (yellow arrowheads). (c) Western blots of MDCK cell extracts for E-cadherin and α-actinin-4 showing resistance to detergent extraction by TX-100. (d) Negative staining of TX-100–treated membranes showing actin filament (red arrowhead) associated with macromolecular complexes (blue arrowhead). (e) Negative staining of CHAPS-treated membranes showing actin filament (red arrowhead) associated with macromolecular complexes (blue arrowhead). (f) Negative staining of TX-100–resistant junctional complex (yellow outlines) showing 6 nm immunogold labeling for α-actinin-4 (yellow arrowheads). (g) Addition of cross-linker–derivatized recombinant α-actinin-4 to high salt stripped membranes results in targeting of α-actinin-4 to a membrane junctional complex that supports actin assembly. Activation of a cross-linker functional group by UV results in covalent attachment of α-actinin-4 to proteins in close proximity. Non-cross-linked proteins are removed by detergent solubilization. Identification of CD2AP, EVL, radixin, and vinculin by mass spectroscopy of gel bands from cleaved α-actinin-4 cross-linked complexes is shown. Coomassie blue staining of SDS-PAGE gel shows purification steps (lanes 1–4).