SMA mutations affect calpain cleavage.

(A, C, E) Illustrative Western blot demonstrating the calpain cleavage susceptibility of several N-terminal mutants. (B, D, F) Quantification of GFP-SMN cleavage was determined from Western blots probed with N-terminal SMN antibodies followed by Cy3 conjugated secondary antibodies (see Methods). The average % of calpain cleavage was calculated from six independent cell-free cleavage assays. Error bars represent the SEM. Asterisks indicate p value, where p<0.005 (*) or p<0.001 (**), determined by two-tailed Student T-test. (A, B) Calpain cleavage of SMN(D30N) was similar to WT, whereas SMN(D44V) was drastically reduced, below the limits of quantification. Therefore, GFP antibodies were used only for detection of the EGFP-SMN(D44V) protein. (C, D) Three mutations within the Tudor domain (I116F, E134K, and Q146Q) showed slightly reduced susceptibility to calpain cleavage, whereas A111G behaved similar to WT. (E, F) Calpain cleavage of A188S was modestly reduced, but its deletion (ΔA188) greatly reduced calpain cleavage, below the limits of quantification.