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Fig. 7. BoNT/B proteolyses Sbr II and SbrIII in rat TGNs but does not reduce K+-evoked CGRP release despite the presence of its receptor; in mouse TGNs, Sbr I is also cleaved and exocytosis is blocked. TGNs were cultured from rat (left panels) and mouse (right panels) for 7 DIV before exposure to BoNT/B; enzyme immuno-assay of CGRP release and western blotting were carried out as described for Fig. 4. Values are the mean ± s.e.m., n=8. (A,C) Immuno-blots showing the disappearance of Sbr isoforms [two (Srb II and Srb III) for rat and three (Srb I, Srb II and Srb III) for mouse] relative to the internal standard (SNAP25) that remained unchanged. (B,D) Dose-response curves for inhibition of CGRP release evoked by 60 mM K+ (
), capsaicin (
) and remaining Sbr II (
), Sbr I and Srb II (
), and Sbr I (
). Inset in D illustrates the inhibition by BoNT/D of K+-evoked CGRP release from mouse TGNs, for comparison. (E,F) Representative fluorescence micrographs showing that the putative protein receptors of BoNT/B, synaptotagmin I and II, are present in CGRP-positive neurons; as well as CGRP and Sbr I are highly colocalized in cell bodies and their fine fibres in rat TGNs. Specimens were stained using (E) rabbit anti-CGRP (1:500) (and donkey anti-rabbit IgG Cy2, 1:200) and goat anti-synaptotagmin I/II (1:100) (and donkey anti-goat IgG Cy3, 1:800) or (F) rabbit anti-Sbr I (1:1000) (and donkey anti-rabbit IgG Cy2, 1:200) and mouse anti-CGRP (1:500) (and donkey anti-mouse IgG Cy3, 1:800). Notice the striking punctate co-staining in the two right hand panels in F. The control was treated in the absence of primary antibodies but incubated with secondary fluorescently labelled IgGs against goat and rabbit. Bars, 20 µm.
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