The presence of WAVE1, WAVE2, and WAVE3 in human platelets. (A-C) Whole cell lysates from platelets (7.5 × 106 cells) or the precipitates eluted from glutathione Sepharose, which had been immobilized with GST or GST-SH3 of abl and then incubated with platelet lysates, were examined for the presence of WAVE1 (A), WAVE2 (B), or WAVE3 (C), as indicated by SDS-PAGE electrophoresis followed by immunoblotting. For WAVE3, 2 polyclonal antibodies were used (see “Materials and methods”). (D) Cos7 cells were transfected with FLAG-tagged WAVE1 to WAVE3 and lysed. The cell lysates (500 μg) were incubated with glutathione Sepharose, which had been immobilized with GST or GST-SH3 of abl. The presence of WAVE1 to WAVE3 was examined as in panels A to C by using an anti-FLAG monoclonal antibody (M2). (E) Whole cell lysates from Cos7 cells expressing similar amounts of FLAG-tagged WAVE1 to WAVE3 or whole-cell lysates from platelets (7.5 × 106 cell) were subjected to SDS-PAGE electrophoresis, followed by immunoblotting as indicated. (Far right panel) FLAG-tagged WAVE proteins were purified with anti-FLAG M2–conjugated agarose, subjected to SDS-PAGE electrophoresis, and stained by CBB. (C1-3 indicates lysates from Cos7 cells expressing FLAG-tagged WAVE1-WAVE3; P, platelet whole-cell lysates). (F) The lysates (20 μg/lane) of Cos7 cells expressing WAVE1 to WAVE3 (C1-3) were subjected to SDS-PAGE, followed by immunoblotting with anti-FLAG (far left panel) or anti-WAVE1 to -WAVE3 as indicated. Mol. Wt. indicates molecular weight in kilodaltons.
