B cell–specific deletion of ERK5 reduces B2 cell numbers. (A) Purified splenic FM B cells from mb1-cre⁺ erk5^fl/fl mice and mb1-cre⁺ erk5^wt/wt control mice were analyzed for ERK5 expression by immunoblotting. (B–F) Flow cytometric analysis of B cell populations in mb1-cre⁺ erk5^wt/wt and mb1-cre⁺ erk5^fl/fl mice from the indicated organs, as shown in Fig. S2. (B) Absolute numbers of total B cells (CD19⁺B220⁺), pro–B (B220⁺CD19⁺IgD⁻IgM⁻CD2⁻), pre-B (B220⁺CD19⁺IgD⁻IgM⁻CD2⁺), immature B (B220⁺CD19⁺IgD⁻IgM⁺CD2⁺), and mature B (B220⁺CD19⁺IgD⁺IgM⁺CD2⁺) cells in the BM (mean ± SEM; n = 7 mice/genotype) were quantified. (C) Absolute splenic numbers (mean ± SEM; n = 14 mice/genotype) of total B cells (IgM⁺ or IgD⁺), immature B cells (B220⁺AA4.1⁺), separated into transitional T1 B cells (IgM^hiCD23⁻) and T2 B cells (IgM^hiCD23⁺) were quantified. Splenic mature B cells (B220⁺AA4.1⁻), separated into FM B cells (IgM⁺CD23⁺) and MZ B cells (IgM^hiCD23⁻). (D) Absolute numbers (mean ± SEM; n = 14 mice/genotype) of B cells (IgM⁺CD19⁺) in peripheral LN (pools of single cervical, axillary, and inguinal nodes; mean ± SEM; n = 14 mice/genotype) were quantified. (E) Proportion of B2 (B220⁺CD19⁺CD5⁻CD23⁺) cells in the peritoneal cavity (mean ± SEM; n = 5 mice/genotype) was quantified. (F) mb1-cre⁺ erk5^wt/wt or mb1-cre⁺ erk5^fl/fl Ly5.2⁺ BM cells were mixed with WT Ly5.1⁺ BM cells at the indicated ratios, and transferred into sublethally irradiated Rag1^−/− mice. After 8-wk reconstitution, absolute numbers of Ly5.2⁺ B2 B cells from LN and spleen were assessed by flow cytometry. Graphs show B2 B cell absolute cell numbers (mean ± SEM; n = 8 independent mice/genotype). Numbers below the graphs represents the ratio between WT Ly5.2⁺ controls compared to ERK5-deficient B cells. In A–F, results are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.