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

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© 2021 Elsevier Inc.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

BACE1 proteolysis of SEZ6, APP, and CHL1 in the hippocampus and olfactory bulb with age.A, representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fractions of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice. Well-characterized BACE1 substrates are also analyzed in the hippocampus and olfactory bulb at three different ages using anti-SEZ6 (14E5; kindly provided by Dr S. Lichtenthaler), anti-CHL1 (AF2147), anti-sAPPβ (BAWT; kindly provided by Dr S. Lichtenthaler), anti-APP (C1/6.1), anti-NCAM1 (AF-2408), anti-NCAM2 (sc-136328), anti-BACE1 (D10E5), anti-GAPDH (MAB374), and anti-calnexin (610523) antibodies. The molecular weight of sSEZ6β in HC (∼155 kDa, arrowhead) is different from the one in the OB (∼165 kDa, double arrowheads) at the age of 4 and 12 months. The weak band at ∼140 kDa (asterisk) represents immature SEZ6-FL, while the strong band at 165-kDa (open arrowhead) or 175-kDa (double open arrowheads) in the OB represents mature SEZ6-FL. The different molecular weight of sSEZ6β or SEZ6-FL between the HC and OB of 4- and 12-month-old mice is most likely due to alternative splicing or posttranslational modifications. In the HC membrane fraction, CHL1-FL was detected at ∼185 kDa at P10, while CHL1-FL is detected as two bands at ∼185 kDa (plain arrow) and ∼175 kDa (dash arrow) in 4- and 12-month-old mice, most likely due to splicing variant or glycosylation. B–G, graphs represent densitometry of soluble protein (sSEZ6β, sAPPβ, and sCHL1) normalized to GAPDH or densitometry of membrane protein (mature SEZ6-FL, APP-FL, and 185-kDa CHL1) normalized to calnexin. White and gray bars represent BACE1+/+ and BACE1−/− mice, respectively. Unpaired two tailed t-test was used for analysis. ∗p < 0.05, ∗∗<0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns; not significant, P10 (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5), 4 months (BACE1+/+; n = 5–6, BACE1−/−; n = 5), 12 months (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5).