LBP-1a is the predominant LSF family member in primary B cells. A) Sequences of LBP-1a (aa 274–336) and LSF (aa 277–332) are aligned. Antipeptide antibodies were produced against nonconserved epitopes (bold). B) Ten ng of His-LBP-1a or His-LSF were subjected to SDS-PAGE and immunoblotted (IB) with the anti-peptide antibodies specific to LBP-1a or LSF, as indicated. C) Extracts (50 µg) from untransfected 293T cells, cells transfected with pCxLSF, or cells transfected with pCxLBP-1a were separated by SDS-PAGE through a 7.5% gel and immunoblotted (IB) with the antipeptide antibodies specific to LSF or LBP-1a, as indicated. D) Left panels: Decreasing levels of purified His-LSF or His-LBP-1a were immunoblotted with the respective antipeptide antibodies. Right panels: on the same gels, whole cell lysates from 20 × 10⁶ splenic mouse B-cells in a single experiment were immunoblotted with antipeptide antibodies specific to either LSF (upper panels) or LBP-1a (lower panels), and subsequently with antibody against β-actin.

LBP-1a binds to Sμ and Sα, but not Sγ1, regions in resting B cells and is released after LPS stimulation. A) Schematic of mouse immunoglobulin S region sequences with predicted LSF/LBP-1a binding sites. Long horizontal lines, to scale, represent each S region sequence; intersecting vertical lines represent predicted LSF/LBP-1a binding sites. Shorter horizontal lines below each S region represent regions amplified in ChIP assays. Amplicon sequences are in boxes below each S region schematic; PCR primers are in bold. Predicted LSF/LBP-1a binding sites within or flanking each amplicon are in shaded boxes. Brackets indicate overlapping sites. B) Isolated primary splenic murine B-lymphocytes were analyzed by ChIP with LBP-1a antiserum. Negative controls included non-specific rabbit IgG (IgG) and preimmune rabbit serum (PI). Input represents 1.25% of ChIP starting material. DNA were amplified with the primers in (A). Data are representative of at least 3 independent experiments. C) Whole cell extracts from 10 × 10⁶ B-cells, stimulated with 50 µg/ml LPS for the indicated amounts of time, were immunoblotted (IB) with antibodies against LBP-1a or β-actin, as indicated. D–E) Resting B-cells were stimulated with 50 µg/ml LPS for the indicated times; LBP-1a occupancy at Sμ (D) and Sα (E) were analyzed as in (B). Upper panel: ChIP analysis with nonspecific rabbit IgG (−) or LBP-1a antiserum (+) at the indicated hours post-LPS stimulation. Lower panel: Amplication of input samples for each time point representing 0.125% (odd lanes) or 0.0125% (even lanes) of the ChIP starting material. Data are representative of 4 independent experiments.

LSFdn expression in B-cells increases switching to IgA, but not to IgG1, upon induction of CSR. A) Isolated B-cells (5 × 10⁶) from LSFdn chimeric mice were sorted into transduced (GFP+) and non-transduced (GFP−) populations based on GFP levels. Cell extracts from a single experiment were immunoblotted for LSF/LSFdn, using the LSF antipeptide antibody (Fig. 1), and for β-actin. B) Efficiency of IgM to IgA switching was determined for splenic B cells isolated from two groups of mice. Control mice (each indicated by a triangle) received bone marrow transduced with parental MSCV retrovirus. LSFdn mice (each indicated by a circle) received bone marrow transduced with MSCV-LSFdn retrovirus. Following stimulation in culture, splenic B cells were simultaneously analyzed for GFP and surface IgA expression. GFP expression levels separated transduced versus nontransduced populations. The fold change in IgA expression upon LSFdn expression, calculated as the ratio of the percentages of transduced cells vs. nontransduced cells that are IgA-positive, is plotted on the y-axis. Left panel: Cells were stimulated with LPS, IL-5, and TGF-β1 (control: n = 5; LSFdn: n = 6). Means (horizontal lines) and SEM were: 1.05 ± 0.17 for control mice, 1.88 ± 0.19 for LSFdn mice. *p=0.0048 (Student’s t-test). Right panel: Cells were stimulated with LPS and IL-4 (control: n = 4; LSFdn: n = 2) Means (horizontal lines) and SEM were: 0.88 ± 0.06 for control mice, 1.43 ± 0.09 for LSFdn mice. The probability that the values in these two data sets would be ordered by chance in this manner is 0.067 (Mann-Whitney U test). C) Splenic B cells derived from control and LSFdn mice characterized in (B) were analyzed for isotype switching to IgG1. Cells were stimulated with LPS and IL-4 (control: n = 6; LSFdn: n = 8). Cells were analyzed for GFP and IgG1 expression, and the fold change in IgG1 expression upon LSFdn expression calculated analogously to above (B). The means and SEM for the ratio of percentage IgG1-positive transduced cells to percentage IgG1-positive non-transduced cells were: 0.98 ± 0.11 for control mice and 0.92 ± 0.14 for LSFdn mice. The difference in means is not statistically significant.