Defective development of pre-B cells in the adult BM of mb.1-Rap1A17 Tg mice. (A) The WEHI231 B-cell line was infected with empty MIG or MIG containing untagged Rap1A17 cDNA. Sorted GFP⁺ cells were stimulated with anti-IgM antibody (20 μg/mL) for 10 minutes, and the lysates were immunoblotted with the indicated antibodies. Rap1GTP and Rap2GTP were assessed by a pull-down assay with GST-RalGDS. (B) A schematic representation of a transgenic Rap1A17^f plasmid (top). MEFs from the Tg mice were infected with empty or Cre-containing adenovirus, Cre-mediated recombination, and Rap1A17 transcripts were assessed by genomic and RT-PCR, respectively, using an indicated primer set. f indicates floxed; Δ, deleted. Rap1GTP was also assessed (bottom). (C) BM cells from mb.1-Rap1A17 Tg mice and Rap1A17^f littermates were multicolor analyzed with the indicated antibodies with the use of FACSCalibur. Cell proportions of the indicated gates are shown. (D) Absolute numbers of each cell fraction in the BM from Rap1A17^f (□) and mb.1-Rap1A17 (■) Tg littermates. Means and SEs of 5 mice are indicated. (E) Cell populations of the indicated phenotypes were sorted from the BM of mb.1-Rap1A17 Tg mice, and Cre-mediated recombination was assessed by genomic PCR.