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. 2014 Jun 30;211(7):1349–1362. doi: 10.1084/jem.20130477

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© 2014 Canault et al.

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Figure 4. — pG248W transition impairs CalDAG-GEFI’s ability to activate Rap1. Activated Rap1 (Rap1-GTP) was detected in platelet lysates obtained from homozygous (HOM) and control (Ctl) subjects at 1 (a) and 5 min (b) after addition of ADP, TRAP-6, or PMA (150 nM). Incubation with GTPγS and GDP were performed on platelet lysates from a homozygous patient. Presented results are representative of three independent experiments. Densitometry analysis shows the band density ratios of Rap1 GTP to total Rap1 as indicated in the panels below. Student’s t test revealed significant differences between control subject and homozygous patient platelet preparation treated with corresponding agonists (*, P > 0.05; **, P > 0.001). Data are mean ± SEM; n = 3. (c) CalDAG-GEFI–dependent activation of Rap1 in HEK293T. Rap1 was co-transfected with either the empty vector or the mutated form of RASGRP2. After stimulation with calcium ionophore (10 µM, 5 min), cells were lysed and Rap1-GTP was pulled down. The representative blots of two independent experiments are shown. Densitometry analysis shows the band density ratios of Rap1 GTP to total Rap1 as indicated in the left panel. Student’s t test revealed significant differences (*, P > 0.05). Data are mean ± SEM; n = 2.