Article Figures & Data
Figures
- Figure 1.
BLyS interacts with NgR. a–c, COS-7 cells transfected with human NgR cDNA are incubated with AP-BLyS (a), with AP-BLyS but precincubated with FLAG-BLyS (b), or with AP (c). Bound AP is visualized using NBT/BCIP as a substrate. d–i, TACI (d), DAF (e), NgRH1 and NgRH2 (f, g), and p75 and Lingo1 (h, i), respectively, are transfected into COS-7 cells. The resulting cells are incubated with AP-BLyS and stained for AP activity visualization. TACI is a known receptor for BLyS. DAF is a GPI-linked protein. NgRH1 and NgRH2 are NgR homolog proteins. p75 and Lingo1 are coreceptors for NgR. Scale bar, 100 μm. j, Quantification of AP-BLyS binding to NgR-expressing COS-7 cells. Binding affinity (Kd) is calculated as 4.60 ± 1.15 nm using the binding saturation curve. Inset is the Scatchard plot. k, Interaction of soluble NgR and BLyS in solution. Purified human NgR-Fc is incubated with human FLAG-BLyS in TBS solution. The mixture was immunoprecipitated with protein A agarose or anti-FLAG M2 beads, respectively. Antibody against FLAG was used to detect FLAG-BLyS. Antibody against human Fc was used to detect NgR-Fc.
- Figure 2.
BLyS is an inhibitor for chicken DRG growth. a, Chicken E12 DRG explants were exposed to 20 nm human Nogo-66 or 16 nm BLyS for 1 h before fixation and stained with Phalloidin, Alexa Fluor 488 nm. Results were analyzed from three independently repeated experiments using Student's one-tailed t test. Scale bar, 50 μm. b, The growth cones' collapse value was increased in the presence of Nogo-66 or BLyS compared with untreated control, as indicated by asterisks (*p < 0.04, **p < 0.009). c, Chicken E12 DRGs were isolated and cultured on immobilized substrates. Sixteen hours after culture, DRGs were fixed and stained with monoclonal antibody Tuj 1 against class III β-tubulin. Scale bar, 100 μm. d, Quantification of E12 chick DRG neurite length inhibition shown in c with Student's two-tailed t test (**p < 0.0001). The number of neurites counted for each treatment is indicated in parentheses. The means ± SEM are presented from four experiments.
- Figure 3.
NgR is indispensable for BLyS inhibition of neurite outgrowth. a, Quantification of E12 chick DRG neurite length on different immobilized substrates. In the presence of NgR-Fc, inhibition of neurite length by BLyS is reduced. **p < 0.001, by Student's two-tailed t test with 95% confidence interval. b, Quantification of DRG neurite length on inhibitory substrates in the presence of PI-PLC. *p < 0.04, by Student's two-tailed t test with 95% confidence interval. c, Quantification of DRG neurite length of wild-type (NgR+/+) and NgR-deficient (NgR−/−) neurons on inhibitory substrates as indicated in d. **p < 0.001, by Student's two-tailed t test with 95% confidence interval. d, P9 DRG neurons from wild-type or NgR-deficient mice were dissociated and cultured on control substrates or substrates containing recombinant Nogo-66 or BLyS. DRGs were fixed and stained using monoclonal antibody Tuj 1 against class III β-tubulin. Scale bar, 100 μm. Neurite length analysis in a, b, and c was performed blindly. The number of neurites counted (means ± SEM) for each treatment is indicated in parentheses.
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