WASP and N-WASP differ in sensitivity to calpain.

WASP was detected with rabbit W485, N-WASP with Ab-1 (D-F) or Ab-2 (B), and μ-calpain with B27D8 monoclonal antibody. (A, B) Intact platelets activated with Ca⁺⁺ ionophore A23187. Note the time-dependent cleavage of WASP, which failed to occur in platelets pretreated with the calpain inhibitors calpeptin23 or E64d24 (A, lanes 5, 6). Note that N-WASP is not cleaved during platelet activation (B); similar results were obtained when blots of platelet lysates were stained with N-WASP Ab-1 (not shown). (C) Platelet sonicates (lysates) supplemented with CaCl₂and incubated at approximately 22°C. Note the conversion of procalpain (80 kd) to calpain (76 kd) and the time-dependent cleavage of WASP, both inhibited by calpeptin. (D) HeLa cell lysates supplemented with CaCl₂ and incubated at approximately 22°C. Note that procalpain was converted to active calpain, but N-WASP was not cleaved over 20 minutes. (E) Combined platelet and HeLa cell lysates (1:1) supplemented with CaCl₂ and calpain (36 μg/mL) (Calbiochem, La Jolla, CA) incubated at approximately 22°C. Note that WASP was cleaved and N-WASP was unaffected. (F) Recombinant N-WASP (700 ng/mL) was added to platelet lysate, supplemented with CaCl₂ and incubated at approximately 22°C.