All mice were maintained on a 12-h lightCdark cycle, and food and water were available ad libitum

All mice were maintained on a 12-h lightCdark cycle, and food and water were available ad libitum. cortex of AD brains (41). Similarly, Leshchyns’ka (15, 17). However, the cleavage of NCAM1 by BACE1 was not previously validated and Table?S1). Consistent with the data obtained with sc-136328 mouse antibody (Figs.?1and S1and and and and and and and and and and are shown in Physique?S4A. ns, Stattic not significant. Next, -secretase processing of NCAM2 was analyzed following the coexpression of NCAM2-Myc-DDK (NCAM2-TM isoform) and BACE1-GFP in HEK cells (Figs.?2 and S4and and and Fig.?S5). Taking in account that this Myc-DDK tag has a weight of 3.6?kDa, the major 34-kDa NCAM1-CTF corresponds to a previously described 30?kDa NCAM1-CTF (47, 49). KCY antibody These fragments are most likely produced by a number of metalloproteinases (46, 47, 48). NCAM1 processing was assessed by quantifying the ratio of the major NCAM1-CTF of 34?kDa (34-kDa NCAM1-CTF) to NCAM1-FL in cell lysates. sNCAM1 levels were measured in a volume of CM adjusted to the protein concentration of the cell lysates. Accordingly, treatment of the cells with either GI or GM resulted in a Stattic reduction of the 34-kDa NCAM1-CTF/NCAM1-FL ratio and secreted soluble fragments of NCAM1 (sNCAM1) in cell lysates and in CM, respectively. In contrast, BACE inhibition (C3) did not produce any change of the 34-kDa NCAM1-CTF/NCAM1-FL ratio or sNCAM1 levels (Fig.?3, and denotes nonspecific bands. GI and GM treatments significantly decreased the levels of NCAM1-CTF (34?kDa) and sNCAM1, indicating that NCAM1 is cleaved by ADAM10 and MMPs. denotes nonspecific bands. and 0.01, ???are shown in Physique?S4B. ns, not significant. Next, -secretase processing of NCAM1 was analyzed following the coexpression of NCAM1-Myc-DDK (NCAM1-140 isoform) and BACE1-GFP in HEK cells (Figs.?3 and S4and S1), indicating that NCAM1 is cleaved by BACE1 at two Stattic different sites in its Stattic ectodomain and and and Fig.?S2and and and and and and (Fig.?4and and and are also analyzed in the HC and OB at three different ages using anti-NCAM1 (AF-2408), anti-NCAM2 (sc-136328), BACE1 (D10E5), anti-calnexin (610523), and anti-GAPDH (MAB374) antibodies. After increasing the contrast of the image for sNCAM1, sNCAM1 was detected in HC and OB of BACE1+/+ mice at P10. Transmembrane (TM) and GPI-anchored (GPI) NCAM2 isoforms were observed in the OB membrane fraction, while only NCAM2-TM was detected in HC membrane fraction. Full-length NCAM1 levels (180, 140, and 120) were observed in membrane fraction. Notably, full-length NCAM1 levels are significantly decreased at P10. and S1increase with age (61), unchanged NCAM1-FL levels in aged BACE1?/? mice could be most likely explained by increased NCAM1 processing by ADAM10. We also observed a weak band of sNCAM1 in the HC of BACE1+/+ mice at P10 after increasing the contrast of the image, and it was significantly reduced in BACE1?/? mice (Fig.?5, and and and and validated BACE1 substrates: SEZ6 (24), APP (63), and CHL1 (18, 19) in BACE1+/+ and BACE1?/? HC and OB of P10, adult, and aged mice. Consistently with a previous study (24), BACE1-specific soluble N-terminal fragment of SEZ6 at 165?kDa (sSEZ6, double arrowheads) was observed in OB soluble fraction of BACE1+/+ mice, but it was almost absent in samples from BACE1?/? mice at all ages (Fig.?6, and and and and and ?and66and lanes 9C10). Subsequent western blot analysis with anti-N-terminal NCAM1 (AF2408) antibody revealed that total NCAM1 levels in BACE1+/+ mice were similar to those in BACE1?/? mice at P10 (Fig.?7and lane 1 lane 2). Next, BACE1 was cotransfected with ST8SIA2 and NCAM1. Ectopic expression of BACE1 produced NCAM1-CTF (38 and45?kDa) in the cell lysates and released sNCAM1 in the CM. Also, we observed an increase in PSA-sNCAM1 levels in the CM, concurrently with a decrease in PSA-NCAM1-FL levels (Fig.?8and and assay. Accordingly, Farzan assay (5). NCAM1 but not NCAM2 is usually a BACE1 substrate in hippocampal synaptosomes of adult mice NCAMs have been shown to regulate formation, maturation, and maintenance of synapses and are enriched in synaptosomes (37, 42, 69). Since BACE1 is also enriched in synaptosomes (18, 70), we prepared hippocampal synaptosomes from 4-month-old BACE+/+ and BACE1?/?.