Fig. 5. The plectin-ABD interacts with other plectin-ABD molecules in vitro and bundles actin filaments. (A) Low-speed sedimentation assay. Polymeric actin (4 µM) was incubated with MBP (2 µM) or MBP-plectin (0.5, 1 or 2 µM) for 1 hour at room temperature. Samples were centrifuged at 14,000 g for 1 minute and equal amounts of pellet (P) and supernatant (S) were subjected to SDS-PAGE and visualized by Coomassie Blue staining. (B) MBP (lanes 1 and 2) and MBP-plectin_1-339 (lanes 3 and 4) bound to amylose-agarose beads were incubated with ³⁵S-labeled plectin-ABD_1-339 (lanes 2 and 4) or ß4^R1281W (lanes 1 and 3), obtained by in vitro translation. After incubation and washing, the beads were boiled in sample buffer and bound proteins were subjected to SDS-PAGE and visualized by autoradiography. Lanes at left show ³⁵S-labeled in vitro-translated plectin-ABD_1-339 and ß4^R1281W. (C) Lysates of COS-7 cells, untransfected (lanes 1, 5, 9) or transiently transfected with HA-plectin-ABD_1-339 (lanes 2, 6, 10), HA-plectin-ABD_65-339 (lanes 3, 7, 11) or HA-ß4^R1281W (lanes 4, 8, 12), were incubated with GST (lanes 5-8) or GST-plectin_1-339 (lanes 9-12), immobilized on glutathione-Sepharose beads. After washing, beads were boiled in sample buffer and associated proteins were visualized by immunoblotting using anti-HA antibody. Lanes 1-4, total COS-7 cell lysates probed by immunoblotting with anti-HA antibody to verify the expression of the HA-tagged proteins. The upper band in lanes 1-4 and 9-10 corresponds to an unidentified protein that is non-specifically recognized by anti-HA antibody.