ELMO1 regulates RhoG activity in the GPVI pathway. (A) Rac1 activation in response to CRP in the absence of ELMO1. Washed platelets were stimulated with 1.5 μg/mL CRP for the indicated times and lysed with NP-40 lysis buffer, and active Rac was pulled down using GST-PAK-RBD. Active Rac1 was detected via western blot analysis using a specific antibody to Rac1. (B-C) Time course of ELMO1^−/− and WT platelets activated with 1.25 μg/mL CRP at the indicated time points under stirring conditions at 37°C. Proteins were precipitated and analyzed by western blot using indicated antibodies. (D) Surface expression of GPVI in washed murine platelets from WT and ELMO1^−/− mice assessed by flow cytometry. GPVI surface expression is a representative of at least 3 independent experiments. (E) RhoG activity in ELMO1^−/− and WT murine platelets in response to CRP. Washed murine platelets were activated with 10 μg/mL CRP for 1 minute and lysed with NP-40 lysis buffer. Active RhoG was pulled down using GST-ELMO1 fusion protein, and RhoG activity was detected by western blot. (F) Model of GPVI signaling by ELMO1 in platelets. All western blot images are representative of at least 3 independent experiments.