Blockade of B cell development and impaired homeostasis in BMKO mice. (A) Southern blot of lysates of thymocytes (Thy) and splenic B cells from Mulefl/fl(y)Mb1Cre (BMKO) mice. The floxed (8 kb) and deleted (6.9 kb) Mule alleles are indicated. (B, left) Immunoblot of purified splenic B cells from control (Mulefl/fl(y)) and BMKO mice detecting Mule protein. β-Tub, loading control. (B, right) Mule expression in control and BMKO pro– and pre–B cells as determined by flow cytometry. (C, left) Flow cytometric analysis of BM from littermate control and BMKO mice showing the pro–B cell (B220lowIgM−cKit+; top) and pre–B cell (B220lowIgM−CD25+; bottom) compartments. Numbers are the percentage of the gated IgM− population. (C, right) Absolute numbers of pro–B cells and pre–B cells in BM isolated from both hind leg femurs (mean ± SD; *, P = 0.006). (D, top) Percentage of apoptotic pre–B cells as determined by flow cytometric analysis of Annexin V expression. (D, bottom) Apoptotic pre–B cells (mean ± SD; *, P = 0.01). Data are from a single experiment involving four to five mice per genotype. (E and F) Flow cytometric analysis of B cell populations in littermate control and BMKO mice. (E, top) Percentage of B220+IgM+ B cells among gated lymphocytes in spleen. (E, bottom) Percentage of CD5+B220+ B1a cells among gated lymphocytes in the peritoneal cavity. Numbers are the percentage among total lymphocytes. (F, left) Splenic B cell cellularity (mean ± SD; ***, P = 1.12−5). (F, right) Percentage of B1a cells in peritoneum (mean ± SD; **, P = 0.0019). Results are representative of two to three (A–C) and three to four (E and F) independent experiments involving one to four mice per genotype.
