Fig. 4. Expression and localization of M. sexta NSF (MsNSF). (A)
Expression of MsNSF is enriched in neuronal tissues of larvae, pupa and
developing adults. Midgut (MG), hindgut (HG) and Malpighian tubules (MT)
contain reduced reactive species. Detergent-solubilized protein extracts (50
µg lane-1) were separated by SDS-PAGE and analyzed by
immunoblotting with affinity-purified 
-MsNSF antibodies. The positions
of molecular mass markers (kDa) are shown. Neonate larvae (B-D) or
fifth-instar midguts (E,F) were stained with -FMRFamide (B,C) or
-MsNSF antibodies (D-F) and detected by Cy3-conjugated secondary
antibodies. Enteric neurons and their axons and a large set of enteric
endocrine cells could be marked by FMRFamide reactivity (B,C). Enriched MsNSF
immunoreactivity (eNSF-IR) is not present in any enteric neurons or their
axons. Six rows of enteric endocrine cells express eNSF-IR only in the median
midgut (D), while staining in regions anterior and posterior was unremarkable
(not shown). Pediculate cells of midgut stain for MsNSF either singly (F) or
in twos (F, inset) per invagination. MsNSF expression predominates in the
basolateral membrane of globet cells (E) and plasma membrane of Malpighian
tubules (G,I). MsNSF expression is also detected in the glomeruli (g; H) in
the antennal lobe of adult and synaptic boutons of larval protocerebrum (J).
(K-M) MsNSF exhibits enrichment in photoreceptors, structures of the optic
lobe and the antennal lobe of M. sexta. MsNSF is expressed in
photoreceptors, mostly localizing to the cytoplasm (M) and granular structures
(K,L) of the optic lobe. Lamina (La), medulla (Me), lobula (Lo) and lobula
plate (Lp) intensely stain for MsNSF, indicating that MsNSF might function in
the visual system of M. sexta. Whole-mount (B-D,J) and paraffin
section histochemistry were performed as described in Materials and
methods.
