Using this assay,

we detected accumulation of ∼90 kDa soluble polypeptide(s) recognized by N-terminal NLG1 antibody in the media (Figure 2B). To control for possible artifacts generated by biotinylation, we filtered media from unbiotinylated neuronal cultures using a 30 kDa cutoff filter, and similar ∼90 kDa bands were detected in the concentrated fractions (Figure S2A). Initially postulated as breakdown products (Ichtchenko et al., 1995), ∼90 kDa NLG1 species have been widely observed and considered to be immature unglycosylated forms of NLG1 (Ko et al., 2009; Scheiffele et al., 2000). To test whether ∼90 kDa NLG1 polypeptide(s) correspond to immature Selleckchem 17-AAG or incompletely glycosylated isoforms, we enzymatically deglycosylated N- and O-linked glycans from media-collected biotinylated fractions and

examined electrophoretic mobility. Deglycosylation resulted click here in equivalent migration shifts of full length and soluble ∼90 kDa NLG1 species (Figure 2C), identifying the latter as cleaved NLG1 N-terminal fragments (NLG1-NTFs) rather than immature nonglycosylated species. To determine if NLG1 is cleaved in vivo, we analyzed soluble fractions of cortical, hippocampal, and cerebellar tissue from adult P60 mice (Figure 2D). Several NLG1 bands of ∼90 kDa were present in all brain fractions, indicating that multiple cleavage fragments are generated in vivo. Enzymatic deglycosylation of these fractions also resulted in a migration shift of NLG1-NTFs, indicating that they originate from mature forms of NLG1 (Figures 2E and S2B). To exclude possible antibody nonspecificity, we tested whether similar bands were detected in extracts of NLG1 KO mice (Varoqueaux et al., 2006). Both 110 kDa full form and ∼90 kDa NLG1-NTFs were absent in NLG1 KO brain extracts and respective soluble fractions (Figure 2F), confirming that the NTFs detected correspond to cleaved NLG1. To characterize the developmental profile of NLG1 cleavage, we performed immunoblot analysis of mouse cortical fractions from birth to adulthood (P1–P60, Figure 2G). Interestingly,

NLG1-NTFs are present throughout development and are enriched during the first postnatal week (P1–P7), where they are as Mephenoxalone abundant as full-length NLG1 (Figures 2G and 2H). A logical outcome of N-terminal proteolysis is the generation of corresponding intracellular C-terminal fragments (CTFs). Analysis of mouse cortical fractions using an antibody targeted against the C-terminal domain of NLG1 revealed multiple membrane-associated ∼20 kDa species, a size consistent with the predicted mass of NLG1-CTFs based on the size of NLG1-NTFs (Figure 2I). These bands are absent in NLG1-KO brain extracts, confirming that they correspond to NLG1 fragments (Figure 2J). To confirm these findings, we expressed a dually tagged version of NLG1 with N-terminal green fluorescent protein (GFP) and C-terminal hemagglutinin (HA) (GFP-NLG1-HA) in COS7 cells.