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Figure 5 The glutathion S-transferase (GST) pull-down competition assays between Tcap and the other Z-disc components. Wild-type (WT) and mutant (R87Q, E132Q, T137I, R153H, S157A, and S157D) Tcap (test-Tcap) and a short Tcap (amino acids 1–120, comp-Tcap) were fused to Thio and T7-tag at the N- and C-terminus, respectively. MLP, titin (Z1-Z2 domain), and calsarcin-1 (CS-1) were fused to GST. The molecular weights of GST-titin, GST-MLP, GST-CS-1, test-Tcap, and comp-Tcap are 50, 47, 39, 35, and 30 kd, respectively. Equivalent amounts of test-Tcap (indicated by arrowheads) and comp-Tcap (indicated by arrows) were mixed with each GST-fusion protein. After incubation and washing, the complex was resolved in SDS-PAGE and immunoblotted with anti-GST antibody and anti-T7 antibody. (a) Input amounts of test-Tcap and comp-Tcap; (b) GST-MLP protein and Tcap proteins in complex; (c) GST-titin protein and Tcap proteins in complex; and (d) GST-CS-1 protein and Tcap proteins in complex.
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