Figure 5.

Secreted immunoglobulin responses by BAFF-responsive NgR1⁺ and NgR3⁺ B-cells are directed against CNS myelin. (a,b) Double immunofluorescence labeling of NgR1⁺ and NgR3⁺ B-cell-secreted IgG (green) co-localized with MBP and Nogo-A, respectively (red), within adjacent spinal cord sections of EAE mice. DAPI (blue) stained cell nuclei. a_i: inset of hatched box in a, b_i: inset of hatched box in b. White arrow indicates co-localization. Scale = 5 μm. (c) Western Immunoblot analysis of secreted IgG in the supernatant from isolated cells. Purified myelin from lumbar spinal cord of ngr1^+/+ and ngr1^−/− mice were differentiated on a 4–12% Bis–Tris gel. IgG presented in the supernatant could directly bind to the major myelin proteins MBP, MOG, PLP and CNPase, annotated in the gel, respectively. The three lanes per box are replicates. No immunoreactive bands were detected when a commercial IgG antibody was utilized as a control.