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. 2000 Feb 21;148(4):653–664. doi: 10.1083/jcb.148.4.653

Figure 3.

Figure 3

Sedimentation assay of myosin assembly. Myosin (1.1 μM) and the 38k protein were mixed in buffer A, and the mixture was allowed to assemble. The assembled myosin was precipitated by centrifugation and quantified. a, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) after centrifugation of the mixture of unphosphorylated myosin and the 38k protein. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). b, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the sedimentation assay with phosphorylated myosin. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). c, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the binding assay of the 38k protein to actin filaments. Actin filaments alone at 10 μM (1 and 2); 10 μM actin filaments and 5.4 μM of the 38k protein (3 and 4); and 10 μM actin filaments, 1.4 μM tropomyosin, and 5.4 μM of the 38k protein (5 and 6). d, The amount of assembled myosin was plotted against concentration of the 38k protein mixed with 1.1 μM myosin. Open and closed circles indicate unphosphorylated and phosphorylated myosin, respectively. e, The amount of the 38k protein bound to myosin was plotted against the 38k protein mixed with 1.1 μM myosin. Open and closed circles denote the unphosphorylated and phosphorylated myosin, respectively. MHC, 38k, A, α-TM, and β-TM denote the bands of MHC, the 38k protein, actin, and α- and β-tropomyosins, respectively.