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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 {alpha}-MsNSF antibodies. The positions of molecular mass markers (kDa) are shown. Neonate larvae (B-D) or fifth-instar midguts (E,F) were stained with {alpha}-FMRFamide (B,C) or {alpha}-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.