Rap1 activation “pulldown” analysis in primary hematopoietic cell populations. (A) Rap1 activation was determined in leukemic cell preparations from 2 B-CLL and 1 T-CLL patient following treatment for 1 hour with 10 μM rolipram/40 μM forskolin (R/F), 300 nM phorbol myristate acetate (PMA), or 50 μM 8CPT-2Me-cAMP using a GST-Ral-GDS pulldown technique (P.D.), followed by anti-Rap1 immunoblotting. As a control, total levels of Rap1 in whole cell lysates (WCL) are shown. (B) Comparable analysis of purified primary human resting or CD40 ligand-activated peripheral blood B cell, T cell, monocyte, and neutrophil populations. All experiments were performed at least twice. The dashed lines in the resting B-cell data indicate that the effect of 8CPT-2Me-cAMP on Rap1 activation was not tested in this particular B-cell sample due to an inadequate number of cells for multiple analyses.