K3 associates with actin and spectrin. (A) Association of K3 with F-actin via cosedimentation. K3 (0.1 μM) or α-actinin (2 μM) or BSA (2 μM) were added to F-actin (1.5 μM) in actin-binding buffer (10 mM Tris-Cl [pH, 7.5], 10 mM NaCl, 50 mM KCl, 2 mM MgCl₂, 0.2 mM CaCl₂, 1 mM adenosine triphosphate) and incubated for 30 minutes at room temperature followed by separation of pellet (P) and supernatant (S) fractions via ultracentrifugation (150 000 g, 90 minutes, room temperature) and analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. (B) Densitometric analysis of samples as described in panel A. Densitometry of gels from 3 different experiments were performed using Image J software and the data are expressed as percentage of K3, α-actinin, or BSA input. (C) Direct binding of purified recombinant K3 to human platelet actin immobilized on 96-well plates; or (D) erythrocyte spectrin. The bound K3 was detected with anti-K3 using enzyme-linked immunosorbent assay, as described in “Materials and Methods.” The results are mean ± SD. (E) K3 (1-105) interacts with actin but not with spectrin and other proteins of junctional complexes. EGFP-tagged WT and mutant K3 as well as control EGFP were overexpressed in K562 cells differentiated with hemin, and immunopurified with EGFP-trap agarose. The immunoprecipitates were analyzed on western blots with the indicated Abs. Three experiments were performed.