Figure 1.
BAFFR-Fc and TACI-Fc, but not Fn14-Fc, induce signals in bone marrow-derived macrophages.
Bone marrow-derived cells from C57BL/6 mice were stimulated for the indicated time with BAFFR-Fc, TACI-Fc or Fn14-Fc at 10 µg/ml, or with LPS at 50 ng/ml. Cells were lysed in sample buffer and analyzed by Western blotting with the indicated antibodies.
Figure 2.
BAFFR-Fc and TACI-Fc, but not Fn14-Fc, induce signals in THP1 and U937 cells, but not in BAFF-expressing 293 cells.
Panel A: THP1 cells were stimulated with BAFFR-Fc, TACI-Fc, Fn14-Fc (all at 5 µg/ml) or LPS (50 ng/ml) for the indicated times. In the P-IκBα blot, bands marked “*” are remnants of the P-ERK blot. The P-IκBα band is just underneath, indicated with an arrow. Bands in the P-ERK blot were quantified, normalized to the intensity of P-ERK at time zero, and plotted as a function of time. Panel B: same as panel A, except that the experiment was performed with U937 cells. Panel C: HEK-293 cells were stimulated with 5 µg/ml of BAFFR-Fc or with 100 nM PMA for the indicated time. Panel D: same as panel C, except that HEK-293 cells stably expressing full-length BAFF [22] were used. In all panels, all extracts were analyzed by western blotting using the indicated antibodies.
Figure 3.
Signalling by BAFFR-Fc and TACI-Fc also takes place in APRIL−/− and BAFF−/− BMDMs, and is not increased in BMDMs with uncleavable BAFF.
Bone marrow-derived macrophages from various mice in the C57BL/6 background were stimulated for the indicated times with BAFFR-Fc, TACI-Fc or Fn14-Fc at 5 µg/ml, or with LPS at 50 ng/ml. Cells were lysed and analyzed by Western blotting with the indicated antibodies. Panel A: wt BMDMs. Panel B: APRIL−/− BMDMs. Panel C: BAFF−/− BMDMs. Panel D: BMDMs from BAFF−/− mice overexpressing non-cleavable BAFF from a BAC transgene [23]. *: as judged by the anti-tubulin blot, less sample was accidentally loaded.
Figure 4.
Gel filtration analysis of BAFFR-Fc, TACI-Fc, Fn14-Fc and atacicept.
Panel A. Superdex-200 elution profiles of the indicated receptors-Fc monitored at 280 nm. The elution position of molecular mass standards is indicated at the top of the profiles. Panel B. BMDMs were treated with TACI-Fc obtained before (total preparation) or after size exclusion chromatography (fraction 8 with high molecular mass aggregates and fraction 14 with dimeric TACI-Fc). Cells were also treated with atacicept (before size fractionation), a form of TACI-Fc unable to bind to Fc receptors. Cell activation was monitored by western blotting with an anti-phospho ERK1/2 antibody. Panel C. BAFF-sensitive BAFFR:Fas expressing cells were stimulated with the indicated concentration of BAFF 60-mer, in the presence or absence of atacicept at a fixed concentration of 300 ng/ml. Cell viability was monitored with a colorimetric assay.
Figure 5.
Impaired TACI-Fc signalling in FcRγ−/− BMDMs.
Bone marrow-derived macrophages from WT and FcRγ−/− C57BL/6 mice were stimulated for the indicated times with TACI-Fc at 10 µg/ml, atacicept at 10 µg/ml, or with LPS at 50 ng/ml. Cell extracts were analyzed by western blotting with the indicated antibodies.