Figure 1

Identification of P-Rex1 as a Rac1-associated protein in platelets. Lysates from quiescent or thrombin stimulated platelets were incubated with glutathione agarose conjugated to GST-tagged Rac1, GDP-loaded Rac1, GTP-loaded Rac1 or GST alone. (A) Captured proteins were eluted into sample buffer and resolved by SDS-PAGE followed by silver staining. (B) Captured protein eluates and whole platelet lysates (input, middle panel) were probed for the presence of P-Rex1 by western blot (WB) with P-Rex1 antisera sc-85805 (Santa Cruz) as previously described [24, 25]. Total Rac1-GST and GST protein inputs for capture experiments are shown by Coomassie stain. (C) Platelets from wild type mice (5 × 10⁸/ml) were treated with 1 U/ml of thrombin for 5 minutes and analyzed for Rac1 activation as previously described [5]. (D) Thrombin-stimulated mouse platelets were lysed in MPER buffer as previously described [19] and incubated with glutathione agarose conjugated to GTP-loaded Rac1-GST or GST alone for 1 hour at 4°C. Eluates were probed for mouse P-Rex1 capture by western blot (WB). Total P-Rex1 from mouse platelet lysates is shown as input (10% of total P-Rex1). Total Rac1-GST and GST protein inputs for capture experiments are shown by Coomassie stain.