Figure 6.

TACI isoforms are associated with activated cleaved TLR9. (A) HEK-293T cells were transfected with either mCherry labeled TACI-S or TACI-L or the S194X mutant (red) along with TLR9-YFP (green); cells were examined by confocal microscopy showing co-localization (yellow) in cells expressing TACI-L or –S, but absent in cells with the S194X mutant. (B) HEK-293T cells were transfected with either TACI-S, TACI-L, or the S194X mutant with TLR9-YFP, to determine complex formation using FRET using FACS (LSRFortessa) as described above. Data is representative of 10 experiments. Right panel shows the % of FRET positive cells in the double positive YFP and mCherry gate in these experiments. Scatter-plot graph shows the mean ± SD. ***p < 0.001, two-tailed paired Student t-test. (C) Lysates of parental BJAB cells or stably transduced with either TACI-S or TACI-L, were examined by immunoblotting using either anti-TACI (top) or anti-TLR9 antibody (bottom) after PAGE gel electrophoresis. Blotting shows both non-activated and activated TLR9 forms (95 and 65kDa) for all cells and TACI isoforms visible only for transduced cells. (D) In other experiments, TACI-S or TACI-L BJAB cells, were incubated for 48 h with APRIL, ODN, or both, were lysed and biotin-labeled goat anti-TACI complexes were collected with SA beads and immunoblotted after PAGE with anti-TLR9 antibody. The activated 65kDa TLR9 cleaved form was predominant in TACI-captured immune complexes (top panel). This effect was enhanced in BJAB-S cells on activation with ODN and even more with ODN + APRIL as activators. Lower panels show TACI and TLR9 expression for the input of these experiments.