293T cells activate TACI signaling in a coculture assay. (A) To detect functional activation by a ligand on 293T cells, TAg Jurkat T cells were transiently transfected with an NFAT-SEAP reporter plasmid and a plasmid directing expression of full-length TACI (TACI), a truncated version lacking the cytoplasmic tail (TACI C-107), or empty vector (control). Equal aliquots of the transfected cells were treated as indicated, with PMA and ionomycin, providing an indication of the maximal activity of the NFAT reporter. For coculture, transfected TAg Jurkat cells were layered onto previously seeded and washed wells containing growing 293T or OV-1063 cells. NFAT-specific SEAP activity was determined and expressed as a fraction of PMA- and ionomycin-induced activity. 293T cells and TACI antibody induced significant activation of NFAT only in cells transfected with full-length TACI expression plasmid. (B) 293T cells do not express either BAFF or APRIL. mRNA was prepared from 293T cells and from known positive cell lines (HeLa for APRIL and HL-60 for BAFF), and RT-PCR was performed as described in “Materials and methods.” 293T cells do not contain detectable mRNA levels of either APRIL (left panel) or BAFF (right panel). (C) A 293T expression library was screened for binding partners with TACI-Fc and revealed syndecan-2 as a candidate. The extent of our syndecan-2 clone is shown here, where the region encoding the signal sequence is highlighted and the area coding for the transmembrane domain is underlined.