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. 2021 Feb 3;296:100372. doi: 10.1016/j.jbc.2021.100372

Figure 1.

Figure 1

NCAM2 and NCAM1 are BACE1 substrates in the mouse olfactory bulb.A–B, schematic diagram of the two mouse NCAM2 (transmembrane [TM] and GPI-anchored) isoforms (A) and three major mouse NCAM1 (transmembrane [NCAM1-180 and NCAM1-140] and GPI-anchored [NCAM1-120]) isoforms (B) resulting from alternative splicing. NCAM2 and NCAM1 have five immunoglobulin (Ig)-like domains represented by ovals and two fibronectin type III repeats (FN) represented by rectangles. C, schematic presentation of NCAM2-TM and polysialylated NCAM1-140 illustrating antibody-binding sites. NCAM2 contains eight putative Asn-linked glycosylation sites (N-glycosylation) indicated by blue triangles, while NCAM1 has six N-glycosylation sites. In the fifth Ig (Ig5) of NCAM1, two of three N-glycosylation sites are polysialylated, represented by a chain of green circles (sialic acid). D, representative immunoblot of PBS soluble fraction (Soluble) and membrane fraction (Membrane) of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using anti-NCAM2 (sc-136328), anti-NCAM1 (AF-2408), BACE1 (D10E5), GAPDH (MAB374), and anti-calnexin (610523) antibodies. BACE1-specific soluble NCAM2 (sNCAM2β) and NCAM1 (sNCAM1β) are observed in BACE1+/+ mice, but not in BACE1−/− mice. Full-length NCAM1 (NCAM1-180 and NCAM1-140, but not NCAM1-120) levels were slightly increased in BACE1−/−, compared with BACE1+/+ mice. However, levels of full-length NCAM2 (NCAM2-TM and NCAM2-GPI) were similar in BACE1+/+ and BACE1−/− mice. E–G, representative immunoblot of membrane fractions of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using two different anti-C-terminal NCAM1 (rabbit pAb; AB5032 [E] and mouse mAb; 0B11 [F]) and anti-C-terminal NCAM2 (goat pAb; GTX89311 [G]) antibodies. After longer exposure, a ∼75 kDa NCAM1-CTF was detected in BACE1+/+ mice, but it was greatly decreased in BACE1−/− mice, and thus termed NCAM1-βCTF (arrow in D and E). However, a BACE1-specific NCAM2-βCTF was not observed (G). H, sagittal section from 12-month-old BACE1+/+ olfactory bulb was stained with anti-BACE1 (3D5, magenta), anti-NCAM2 (AF778, green), and anti-NCAM1 (AB5032, red) antibodies. NCAM1 and NCAM2 immunostaining with BACE1 are significantly overlapped in glomeruli. Scale bar represents 100 μm. D (BACE1+/+; n = 5–6, BACE1−/−; n = 5); E–G (BACE1+/+; n = 3 and BACE1−/−; n = 3); H (BACE1+/+; n = 4, two sagittal (H) and two coronal [Fig. S3] sections).