In vitro binding of GST fusion proteins to WAVEs and WASP. (A-D) Platelet lysates prepared without stimulation were subjected to GST pull-down assay. The precipitates eluted from glutathione Sepharose, which had been immobilized with GST, GST–profilin I, GST-SH3 domain of IRSp53, or GST-SH3 (the amino terminal SH3) of CrkL, were equally divided and examined for the presence of WAVE1 (A), WAVE2 (B), WASP (C), or WAVE3 (D) as indicated by immunoblotting. For WAVE3 (D), whole-cell lysates from platelets (7.5 × 10⁶ cells) were also subjected to SDS-PAGE followed by immunoblotting. (E-G) Cos7 cells were transfected with FLAG-tagged WAVE1 (E), WAVE2 (F), or WAVE3 (G) and lysed. The cell lysates (500 μg) were incubated with glutathione Sepharose, which had been immobilized with GST, GST-SH3 of abl, GST–profilin I, GST-SH3 domain of IRSp53, or GST-SH3 (the amino terminal SH3) of CrkL as indicated. The presence of WAVE1 to WAVE3 was examined by using an anti-FLAG monoclonal antibody (M2).