GTPase activity of Rap1GAP2. COS-1 cells were transiently transfected with HA-tagged Rap1B and VSV-tagged CalDAG-GEFIII (CD-GEF III). In addition, increasing amounts of VSV-tagged Rap1GAP2a or HA-tagged Rap1GAP1 were expressed as indicated. Two days after transfection, cells were lysed, and pull-down assays with an activation-specific probe were performed to determine the amounts of Rap1B-GTP (A). Levels of total Rap1B, Rap1GAP2, Rap1GAP1, and CalDAG-GEFIII were determined with tag-specific antibodies. Blots from 4 independent pull-down experiments were scanned and quantified (B). To compensate for differences in total Rap1 expression levels, ratios of Rap1-GTP to total Rap1 signals were calculated. Shown are mean ± SEM. Rap1GAP2 and Rap1GAP1 blocked CalDAG-GEFIII-induced activation of Rap1B with similar potency.