1. LAT Links the Pre-BCR to Calcium Signaling
Yu-wen Su, Hassan Jumaa 
Immunity 
Volume 19, Issue 2, Pages (August 2003)
DOI: /S (03)

2. Figure 1 Expression of LAT and SLP-76 in pre-B Cells
(A) Total cellular lysates from BM-derived pre-B cells and various B cell lines (as indicated in the figure) were subjected to immunoblotting with anti-LAT (upper panel), anti-SLP-76 (middle panel), or, as a control, anti-eIF-4A (lower panel). The murine T cell line RLM-11 was used as positive control for LAT and SLP-76 expression (lane 8).
(B) B220 versus μ FACS profiles of the BM-derived pre-B cells from WT, SLP-65−/−, and SLP-65/LAT−/− mice used in (A).
(C) LAT and SLP-76 are tyrosine phosphorylated upon pre-BCR engagement. Immunopurification with anti-LAT or anti-SLP-76 from total cellular lysates of SLP-65−/− pre-B cells. Cells were either left untreated (−, lanes 1 and 3) or stimulated with anti-μ (+, lanes 2 and 4) at 37°C for 2 min. Membranes were incubated with anti-pY (upper panel) and then reblotted with anti-LAT and anti-SLP-76, respectively (lower panel).
Immunity  , DOI: ( /S (03) )

3. Figure 2 B Cell Development in SLP-65/LAT−/− Mice
(A) Analysis of B220 and CD43 expression was carried out for lymphocytes in the BM (left) and spleen (right) of the indicated mice. Numbers refer to the percentage of cells in the rectangle relative to blotted cells.
(B) Analysis of pre-BCR expression (μ versus λ5) was carried out for B220+ cells in the BM. Numbers refer to the percentage of cells in the gate relative to blotted cells. Analysis of IgM versus IgD expression was carried out for B220+ cells in the spleen. Numbers refer to the percentage of cells in the quadrants relative to blotted cells.
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4. Figure 3 BM Transfer to Reconstitute B Cell Development in μMT Mice
μMT mice were irradiated and subsequently injected either with PBS, BM cells from WT, SLP-65−/−, or SLP-65/LAT−/− mice. BM cells from two donor mice of each genotype were combined prior to injection. The lymphocytes in BM and spleen of the injected mice were analyzed for B cells by flow cytometry (B220 versus IgM and IgD versus IgM). Five different μMT mice were used for each genotype with similar results.
Immunity  , DOI: ( /S (03) )

5. Figure 4
Correlation of Ca2+ Mobilization with LAT Expression in SLP-65−/− pre-B Cells
(A) Total cellular lysates from the pre-B cell lines Dec, Oct, and 84 were subjected to LAT immunoblotting (upper panel) followed by reblotting for eIF-4A as control (lower panel).
(B) SLP-65−/− pre-B cells that express LAT show Ca2+ mobilization upon pre-BCR engagement. Ca2+ mobilization profiles after anti-μ treatment of the indicated cell lines are shown.
(C) Increased association of LAT with PLC-γ2 in Dec pre-B cell line upon pre-BCR engagement. Immunopurification with anti-LAT from total cellular lysate of Dec cells left unstimulated (−) or stimulated with anti-μ (+) at 37°C for 2 min. The membrane was incubated with anti-PLC-γ2 (upper panel) followed by anti-LAT (lower panel).
(D) Phosphorylation of Ig-α upon pre-BCR engagement. Immunopurification with anti-pY from the cellular lysates of Dec and 84 pre-B cells unstimulated (−) or stimulated with anti-μ (+) for 2 min. The membrane was incubated with anti-pY (upper panel) followed by reblotting for Ig-α (lower panel). The position of tyrosine phosphorylated Ig-α is indicated.
Immunity  , DOI: ( /S (03) )

6. Figure 5
LAT and SLP-76 Form a Constitutive Complex in pre-B Cells that Is Recruited to the pre-BCR Upon Engagement
(A) Immunopurifications with anti-LAT from total cellular lysates of SLP-65−/− Dec pre-B cell line and wt2 (a BM-derived pre-B cell line from a wild-type mouse). The cells were left untreated (−) or stimulated with anti-μ (+) at 37°C for 2 min. The membrane was incubated with anti-pY (upper panel) followed by reblotting with anti-Ig-α (middle panel) and anti-LAT (lower panel).
(B) Expression of SLP-65 in wt2 revealed by Western blot of total lysates and CD19 versus μ FACS profiles of the pre-B cell lines wt2 and Dec.
(C) Immunopurifications with anti-SLP-76 from total cellular lysates of Dec and wt2. The cells were left untreated (−) or stimulated with anti-μ (+) at 37°C for 2 min. The membrane was incubated with anti-pY (upper panel) followed by reblotting with Ig-α (middle panel) and anti-SLP-76 (lower panel).
(D) Constitutive association of LAT with SLP-76 in pre-B cells. Dec cells were left untreated (−) (lanes 1 and 4), stimulated with anti-μ for 2 min (μ, lanes 2 and 5) or with pervanadate for 3 min (pv, lanes 3 and 6). Immunopurifications with anti-SLP-76 and anti-LAT from cellular lysate were dephosphorylated and subjected to immunoblotting with anti-SLP-76 (upper panel) and anti-LAT (lower panel). Similar results were obtained with wt2 pre-B cells.
Immunity  , DOI: ( /S (03) )

7. Figure 6 Similar Functions for LAT and SLP-65 in pre-BCR Signaling
(A) Schemes of fusion proteins between LAT and GFP and confocal microscopy photographs to show the distribution of the GFP-fusion proteins in pre-B cells. LAT-GFP fusion protein was expressed on the cell surface (upper) but not the GFP-LAT fusion protein (lower).
(B) Expression of LAT-GFP in pre-B cells. Pre-B cells 84 were transduced with the retroviral construct encoding LAT-GFP. 24 hr later, the transduction efficiency was determined (30%) and the expression of LAT-GFP was analyzed by anti-LAT immunoblot of total lysates (upper panel). The membrane was reblotted with anti-eIF4-A (lower panel). The positions of LAT and LAT-GFP are indicated.
(C) Expression of GFP-SLP-65 in pre-B cells. Pre-B cells 84 were transduced with the retroviral construct encoding GFP-SLP hr later, the transduction efficiency was determined (40%) and the expression of GFP-SLP-65 was analyzed by anti-SLP-65 immunoblot of total lysates (upper panel). The membrane was reblotted with anti-eIF4-A (lower panel). The positions of SLP-65 and GFP-SLP-65 are indicated.
(D) GFP versus μ FACS profiles and μ histograms showing LAT-induced downregulation of the pre-BCR. SLP-65/LAT−/− pre-B cells (84) were transduced either with LAT-GFP, GFP-LAT, GFP-SLP-65, or GFP. 24 hr after transduction, the cells were stained with cy5-labeled anti-μ and analyzed by FACS. The GFP-negative and GFP-positive populations were gated as R1 and R2, respectively. Pre-BCR downregulation is shown in histograms comparing μ expression between R1 and R2.
(E) Ca2+ mobilization upon pre-BCR engagement of the transduced cells described above. 24 hr after transduction, cells were stimulated with anti-μ and Ca2+ response was compared between cells in R1 and R2 of the same sample.
(F) LAT induces pre-B cell differentiation. After 4 days culture in medium lacking IL-7, the cells described in (D) were analyzed for κ expression. The numbers refer to the percentages of differentiated cells relative to total cells of each histogram.
Immunity  , DOI: ( /S (03) )

8. Figure 7
Model for the Mechanisms of PLC-γ2 Activation by pre-BCR Signaling
Phosphorylated and pre-BCR associated SLP-65 provides docking sites for PLC-γ2 and Btk (Tec kinase) resulting in full activation of PLC-γ2 and Ca2+ mobilization. In an alternative complex, SLP-76 may bind to Ig-α and become phosphorylated by Syk. SLP-76 interacts via the adaptor proteins Gads or Grb2 with LAT that can also be phosphorylated by Syk. Phosphorylated LAT recruits PLC-γ2 to the pre-BCR, resulting in PLC-γ2 activation and Ca2+ mobilization.
Immunity  , DOI: ( /S (03) )