Calpain-mediated cleavage of WAVEs. (A-C) Platelets were treated with calpeptin (20 μM) (lane 3) or 0.1% dimethyl sulfoxide (vehicle for calpeptin; lanes R = resting and D = dibucaine) for 15 minutes. The cells were then lysed in 1% SDS, before or 15 minutes after the addition of dibucaine (1 mM) or dibucaine + calpeptin. The proteins from 1.5 × 106 cells were separated on SDS-PAGE. After transferring to the nitrocellulose membrane, the membrane was probed with isoform-specific anti-WAVE antibodies. Lane R indicates resting cells; lane D, dibucaine-treated cells; and lane DC, calpeptin- and dibucaine-treated cells. (D-F) In vitro cleavage of WAVEs. WAVE1 or WAVE 2 was precipitated from platelets either by GST–profilin I (for WAVE1) or GST-IRSp53 (for WAVE2) as indicated. FLAG-tagged WAVE3 was expressed in Cos7 cells and immunoprecipitated by anti-FLAG monoclonal antibody. The precipitates were untreated or incubated with purified μ-calpain in the presence or absence of calcium ion as indicated. After denaturing in SDS, WAVE1 (D), WAVE2 (E), and WAVE3 (F) were detected by immunoblotting. (G) Cleavage of WAVEs during platelet aggregation. Platelets were stimulated with thrombin (1 U/mL) for 30 minutes with or without stirring. After the addition of EGTA (5 mM) and ethylenediaminetetraacetic acid (5 mM), platelets were lysed by boiling in SDS sample buffer. WAVEs were detected by immunoblotting as described in panels A to C. Lane 1 indicates resting platelets; lane 2, thrombin stimulation of platelets for 30 minutes with stirring; and lane 3, thrombin stimulation for 30 minutes without stirring.
