SH3 domains of CrkL and PSTPIP1 bind to WASP in vitro.

(A) Platelet lysates prepared with or without collagen stimulation were subjected to GST-CrkL pull-down assay. Precipitates eluted from glutathione Sepharose, which had immobilized GST, GST-CrkL-SH2, or GST-SH3 (the amino terminal SH3), were examined for the presence of CrkL. (B) The same as in panel A, except that GST-SH3 domain of PSTPIP1 or beads alone were used. (C) Direct binding of PSTPIP1 to immobilized WASP. Platelet lysates prepared before and after stimulation with collagen were divided in half. Anti-WASP or preimmune serum was added. Precipitates were then divided in half, separated by 7.5% to 15% SDS-PAGE, and transferred to nitrocellulose membrane. Membranes were probed with the GST-SH3 domain of PSTPIP1 or with anti-WASP. To detect the membrane-bound GST fusion proteins, membranes were further probed with anti-GST. HC indicates heavy chains of antibodies.