Igβ_c→α_c mice. (A) Scheme for gene targeting depicts the genomic Igβ locus (top), targeting vector (middle), and recombinant allele after neomycin gene deletion (bottom). Exons are represented by boxes (Igβ cytoplasmic exons [black] and Igα cytoplasmic exons [crosshatched]), lox P sites by open triangles, and the probe is shown by a striped box. (B) Western blot analysis. Wild-type (wt) and Igβ_c→α_c B cell lysates were probed with antibodies specific for the cytoplasmic domains of Igα and Igβ. Numbers above the lanes represent micrograms of lysate per sample. Bands with the expected relative mobility of Igα, Igβ, and Igβ_c→α_c proteins are indicated to the left of the figure. (C) B cell development in the bone marrow of wild-type, IgβΔC, and Igβ_c→α_c mice. Numbers represent the percentages of lymphocytes except for the plots depicting CD25 versus IgM that were gated on B220⁺ cells. (D) Spleen B cells in wild-type, IgβΔC, and Igβ_c→α_c mice. Numbers represent the percentages of lymphocytes. Rows 2 through 4 were pregated on the B220⁺ population. Transitional 1 cells (T1) were HSA⁺CD21⁻, transitional 2 cells (T2) were HSA⁺CD21⁺, and mature (M) B cells were HSA^loCD21⁺. Peritoneal B cells were gated on lymphocytes as per forward versus side scatter parameters and then fractionated into B1a (B220^lo CD5⁺), B1b (B220^lo CD5⁻), and B2 (B220^hiCD5⁻).

Cell surface BCR expression. (A) IgM and IgD surface expression on B cells from bone marrow and spleen of wild-type, Igβ_c→α_c, Igβ_c→α_c/Igα^FF/FF, and Igα^FF/FF mice. Splenic B cells were purified by negative selection before staining. (B) IgM internalization assessed by flow cytometry. Percent internalization was calculated by the formula % sIgM(T₀) − % sIgM(T_n)/% IgM(T₀) × 100. The graph shows three separate experiments. Wild type (filled diamonds) and Igβ_c→α_c (filled squares).

Antibody responses in Igβ_c→α_c mice. Graphs show anti-NP IgM and IgG responses measured by ELISA on days 7, 14, 21, and 28 (for NP-CGG) and days 7, 14, 21, and 31 for (NP-Ficoll) after immunization with T-dependent (NP-CGG) or -independent (NP-Ficoll) antigens. The filled diamonds and the open squares represent serum titers in individual wild-type and Igβ_c→α_c mice, respectively. The ordinate shows OD 415 nm relative to nonimmunized controls. The line shows the means for each group at a given time point. There were three mice in each group.

Protein tyrosine phosphorylation. Purified B cells were stimulated with 10 μg/ml goat anti–mouse F(ab)′₂ for the indicated times. (A) Tyrosine phosphorylation of total cellular proteins (5 × 10⁶ cells/lane) detected by antiphosphotyrosine immunoblotting with the mAb 4G10. (B) Tyrosine phosphorylation in Igα immunoprecipitates. Igα immunoprecipitates were immunoblotted with mAb 4G10 to detect tyrosine phosphorylation. The lower panel shows the same blot stripped and reprobed with Igα antibody. (C) Syk tyrosine phosphorylation. Anti-Syk immunoprecipitates were immunoblotted with mAb 4G10 to detect tyrosine phosphorylation. The lower panel shows the same blot stripped and reprobed with anti-Syk antibody.

B cells in bone marrow and spleen in wild-type and Igβ_c→α_c mice. Numbers show percentages of bone marrow or splenic lymphocytes. (A) Bone marrow. Pro B cells were CD43⁺B220⁺, pre BII cells were CD25⁺IgM⁻, immature B cells were IgM⁺IgD^lo, and mature B cells were IgM⁺IgD^hi. (B) Spleen. T1, T2, and mature B cells were defined as in Fig. 1. Marginal zone B cells were CD21⁺CD23⁻ and follicular B cells were CD21⁺CD23⁺. Filled diamonds represent wild-type and open squares represent Igβ_c→α_c mice. Each symbol shows an individual mouse. Bars represent the means of all mice in a group.

(A) B cell lifespan in bone marrow and spleen in wild-type and Igβ_c→α_c mice measured by BrdU. Diamonds show wild-type B cells and circles show the Igβ_c→α_c B cells. Immature B cells (bone marrow) and mature B cells (spleen) were defined as in previous figures. Half-life was the time when 50% of the cells were BrdU labeled. Immature B cells were as in previous figures. Spleen immature B cells were IgM⁺IgD^lo, mature B cells were IgM^loIgD^hi, and T cells were CD3⁺.

In vitro responses to mitogens. (A) Histograms show CFSE staining on wild-type, Igβ_c→α_c, Igα^FF/FF, and Igβ_c→α_c/Igα^FF/FF CD19⁺ cells cultured with Fab′2 anti-IgM. The dark line represents cells at time 0 with no stimulation, the light line represents number of cell divisions after 72 h in culture. (B) Up-regulation of activation markers in wild-type and Igβ_c→α_c B cells. Cell surface expression of CD69 and CD86 after 24 h incubation with 10 mg/ml goat anti–mouse (Fab′)₂ fragments or CD40L. Solid peaks show unstimulated cells and open peaks represent expression after stimulation. (C) [³H]Thymidine incorporation by wild-type (black bars) and Igβ_c→α_c (hatched bars) B cells after a 54-h culture. Error bars show means on triplicate cultures in an experiment representative of three independent experiments. (D) Histograms show surface expression of CD40 and RP105 by wild-type (orange) and Igβ_c→α_c (green) B cells. (E) Histograms show CFSE dilution by wild-type (orange) and Igβ_c→α_c (green) CD19⁺ cells stimulated with CD40L, or CpG, or LPS, or RP105 after a 72-h culture. (F) Ca²⁺ flux response to BCR cross-linking in splenic B cells in wild-type and Igβ_c→α_c mice. Contour plots represent Ca²⁺ flux of splenic B cells measured by the fluorescence 395:510 nm ratio of Indo-1-AM emission accumulated over 1,024 s. Splenic B cells were gated by anti-B220 and anti-Fab′ IgM staining. Baseline fluorescence was acquired for 60 s before cross-linking with goat anti–mouse IgM (Fab′)₂ at a final concentration of 30 μg/ml.