Fig. 2. Biochemical analysis of plectin/actin interactions. (A) Copolymerization assays were performed using bovine -skeletal muscle actin at 2.5 µM together with different concentrations of MBP-plectin-ABD_1-339 (lanes 1-8 correspond to 0.2, 0.37, 0.5, 0.6, 1, 1.5, 1.6 and 2.2 µM, respectively). Actin filaments and bound proteins were sedimented by centrifugation and equivalent samples of pellets (upper panel) and supernatants (lower panel) were resolved by SDS-PAGE. (B) Scatchard plot of plectin binding to actin filaments (values are means differing by less than 5% of duplicate experiments). Scatchard plotting indicates that plectin binds to filamentous actin with an apparent K_d of 0.3 µM and a molecular ratio of 1 plectin molecule per actin monomer. (C) Pull-down assay of plectin-ABD_1-339/G-actin binding. Immobilized MBP-plectin-ABD_1-339 in suspension (3 µM) was incubated with soluble -skeletal muscle G-actin (2.5 µM) in the absence (lanes 1 and 2) or presence of a fivefold excess of soluble MBP-plectin-ABD_1-339 (lanes 3 and 4). After incubation, the beads were pelleted by centrifugation and the supernatants removed. The beads were then washed and the proteins eluted by boiling in sample buffer. Equivalent samples of supernatant (unbound (UB) actin, lanes 1 and 3) and bead eluates (bound (B) actin, lanes 2 and 4) were analyzed by SDS-PAGE. Proteins were visualized by Coomassie Brilliant Blue staining.