Tmod3 and -4 bind to striated muscle TMs more weakly than does Tmod1. (A) Increasing amounts of Tmods were separated by SDS-PAGE, transferred to nitrocellulose, and overlaid with wild-type (WT) αslow-TM (first row), M9R mutant αslow-TM (second row), or β-TM (third row). (B) Increasing amounts of TMs were separated by SDS-PAGE, transferred to nitrocellulose, and overlaid with Tmod1 (first row), -4 (second row), or -3 (third row). Parallel Coomassie-stained gels show loading (fourth row). (A and B) Molecular mass is indicated in kilodaltons. (C) Relative binding strengths among Tmods and TMs were scored semiquantitatively based on visual inspection of blots. Predicted binding between Tmods and αfast-TM (parentheses) is based on sequence identity between the N-terminal regions of αfast-TM and β-TM where Tmods bind (Stone and Smillie, 1978; Helfman et al., 1986). Asterisks indicate conserved residues. The arrow shows the location of the nemaline myopathy–causing M9R mutation. (D and E) Binding avidities were quantified using densitometry by normalizing band intensities from the blots to the corresponding Coomassie-stained bands. x-axis labels refer to the protein that is overlaid, whereas data series refer to the immobilized protein. Note that relative binding avidities can only be compared within each individual blot overlay, demarcated by tick marks, and not from one TM to the next (in D) or from one Tmod isoform to the next (in E) because of variations in antibody binding and blot exposure times. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = 4 lanes/group. Data are mean ± SEM. a.u., arbitrary unit.
