Expression and purification of Flag-sTALL-1. (a) Expression of Flag-sTALL-1. 293 cells were transfected with pSec-Flag-sTALL-1 or an empty control plasmid. Twenty-four hours after transfection, cell culture medium was collected and immunoprecipitated with a monoclonal anti-Flag antibody (lanes 2 and 4) or mouse IgG control (lanes 1 and 3). The immunoprecipitates were analyzed by Western blot with anti-Flag antibody. (b) Purification of Flag-sTALL-1. Conditioned medium from a 293 stable cell line expressing Flag-sTALL-1 was passed through an anti-Flag affinity column. Bound proteins were eluted with Flag peptide and analyzed by Coomassie blue staining (left lane) or Western blot with anti-Flag antibody (right lane).

Specific interaction between BCMA and TALL-1. (a) BCMA interacts with sTALL-1 but not sTRAIL. 293 cells were transfected with pCMV-BCMA-HA (lanes 1–4, 7, and 8) or an empty control plasmid (lanes 5 and 6). Twenty-four hours after transfection, cells were lysed and the lysates were mixed with control medium (lanes 1 and 2) or conditioned medium containing Flag-sTALL-1 (lanes 3–6) or Flag-sTRAIL (lanes 7 and 8). The mixtures were immunoprecipitated with a monoclonal anti-HA antibody (lanes 2, 4, 6, and 8) or mouse control IgG (lanes 1, 3, 5, and 7). The immunoprecipitates were analyzed by Western blot with anti-Flag antibody (Upper). The same blot was stripped and reprobed with anti-HA antibody (Lower). The higher-molecular-weight band is probably a glycosylated form of BCMA, which has been shown to be a glycoprotein (25). (b) sTALL-1 does not bind to TRAIL receptors. 293 cells were transfected with secretion expression plasmids for Flag-Fc, Flag-sTRAIL-R1-Fc, and Flag-sTRAIL-R2-Fc. Twenty-four hours after transfection, aliquots of the cell culture medium were mixed with conditioned medium containing either Flag-sTALL-1 (lanes 1–3) or Flag-sTRAIL (lanes 4–6). The mixtures were precipitated with GammaBind G Sepharose beads. Proteins bound to the beads were analyzed by Western blot with anti-Flag antibody.

Flow cytometry analysis of sTALL-1 binding to BCMA. (a) TALL-1 receptor expression in various cell lines detected by Flag-sTALL-1. The indicated cells were incubated with control medium (shaded histograms) or Flag-sTALL-1 containing medium (solid-line histograms). Cells were washed and analyzed by flow cytometry with a monoclonal anti-Flag antibody. (b) BCMA is targeted to plasma membrane, where it can bind to sBCMA. 293 cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cells were incubated with control medium (shaded histograms) or Flag-sTALL-1 containing medium (solid-line histograms). Flow cytometry analysis of the transfected intact cells was performed with anti-Flag antibody. (c) sBCMA blocks sTALL-1 binding to its receptor. Bjab cells were incubated with control medium (shaded histogram), Flag-sTALL-1 containing medium + control medium (solid-line histogram), or Flag-sTALL-1 containing medium + Flag-sBCMA-Fc containing medium (dashed-line histogram). The treated cells were analyzed by flow cytometry with anti-Flag antibody. Similar data have been obtained for the above flow cytometry analysis with purified Flag-sTALL-1.

Inhibition of TALL-1-triggered B cell costimulation by sBCMA. Purified B lymphocytes cultured in 96-well dishes were treated for 40 h with the following reagents: (i) TBS control buffer, (ii) Flag-sTALL-1 (100 ng/ml), (iii) anti-IgM (10 μg/ml), (iv) Flag-sTALL-1 (100 ng/ml) + anti-IgM (10 μg/ml), (v) Flag-sTALL-1 (100 ng/ml) + anti-IgM (10 μg/ml) + concentrated Flag-sBCMA-Fc (5 μl), and (vi) Flag-sTALL-1 (100 ng/ml) + anti-IgM (10 μg/ml) + concentrated Flag-Fc (5 μl). The treated cells were pulsed with [³H]thymidine for 10 h. [³H]Thymidine incorporation was measured by liquid scintillation counting.

BCMA activates NF-κB, and the activation is potentiated by sTALL-1. 293 cells were transfected with 0.5 μg of NF-κB luciferase reporter plasmid and increased amounts of an expression plasmid for BCMA. Fourteen hours after transfection, cells were treated with 100 ng/ml Flag-sTALL-1 (solid bars) or left untreated (open bars) for 7 h and luciferase reporter assays were performed.

TRAF5, TRAF6, NIK, and IKKs are involved in BCMA-mediated NF-κB activation. (a) Effects of various dominant negative mutants on BCMA-mediated NF-κB activation. 293 cells were transfected with 0.5 μg of NF-κB reporter plasmid, 0.5 μg of BCMA expression plasmid, 1 μg of control plasmid, or expression plasmids for the indicated mutants. crmA plasmid (0.5 μg) also was added to the RIP () transfections for inhibiting apoptosis. Twenty hours after transfection, luciferase assays were performed and data were treated as described in Materials and Methods. Expression of the HA-tagged BCMA and the Flag- or Myc-tagged mutants was examined by Western blot analysis with antibodies against the HA, Flag, or Myc epitopes, respectively. TRADD(296S) and IκBα(SS/AA) are not tagged and could not be detected in these experiments. (b) BCMA interacts with TRAF5 and TRAF6 but not TRAF2. 293 cells were transfected with expression plasmids for HA-BCMA and the indicated plasmids. Transfected cell lysates were immunoprecipitated with anti-Flag antibody or control IgG. The immunoprecipitates were analyzed by Western blot with anti-HA antibody (Top). Expression of the transfected proteins was detected by Western blot analysis with anti-Flag (Middle) or anti-HA antibody (Bottom).