Inhibition of neural NO-synthase diminishes release of AM-containing HS (HS-anMan) from glypican-1 (Gpc-1), prevents TNF-α-induced clustering of α-SYNfils in SH-SY5Y cells and increases α-SYN secretion. Representative immunofluorescence images of SH-SY5Y cells which were grown to confluence in regular medium (A, C; UT = untreated), or in medium containing 100 μM S-methyl-L-thiocitrulline (B, D; SMTC) or 100 pg/ml tumor necrosis factor-α (E, TNF-α) or both (F, TNF-α + SMTC). Staining was performed with mAb AM (for HS-anMan, green), pAb SYN (red), mAb SYNfil (green) and DAPI (for nuclei, blue). Exposure time was the same in all cases. Bar, 20 μm. (G) Slot blot assay of SYN secretion into the media (total volume, 2.8 ml) of cell cultures grown to confluence in the presence of TNF-α or TNF-α and SMTC. Sample volume: 30 μL. The intensity of the controls (no primary antibody added) was subtracted. The error bars indicate S.E., n = 5; ****, P < 0.0001.