Fig 1: DPV of varying BACE 1 Ag concentrations in buffer samples: (Ai). testing of varying BACE1 Ag concentrations ranging from 1 μM to 1fM at 10-fold dilutions; (Aii). standard calibration graph of log of BACE1 Ag concentrations in buffer vs. I/μA = peak current/blank current; DPV of varying BACE 1 Ag concentrations in spiked serum samples: (Bi). testing of varying BACE1 Ag concentrations ranging from 1 μM to 1fM at 10-fold dilutions; (Bii). standard calibration graph of log of BACE1 Ag concentrations in serum vs. I/μA = peak current/blank current.
Fig 2: rGO synthesis and conjugation characterization: (A) FT-IR spectrum of GO shows peaks at 1,630 cm−1 (C = C stretching) and 1,070 cm−1 (C-O stretching) whereas rGO shows only a single peak at 160 cm−1 (C = C stretching); (B) Raman graph showing a higher G-band than D-band by 10 a.u. intensity for GO but no such difference for rGO; (C) EDX spectrum showing (Ci). higher amount of oxygen in GO and (Cii). lower amount of oxygen in rGO; (D) scanning electron micrographs of (Di). rGO, (Dii). BACE1 Ab deposited on rGO, (Diii). BACE1 Ag bound to BACE1Ab on rGO.
Fig 3: Differential spatiotemporal BACE1 processing of NCAM1 and NCAM2 in vivo.A, representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fraction 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 in vivo 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, sNCAM1β 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. B–I, graphs represent densitometry of soluble protein normalized to GAPDH or densitometry of membrane protein 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, N.A.; not applicable, 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). Full-length versions of the western blots (sNCAM2β and sNCAM1β) of 4-months-old mice shown in Figure S1, A and C.
Fig 4: NCAM2 is cleaved by metalloproteinases or BACE1, followed by γ-secretase in HEK cells. HEK cells were transfected with NCAM2-Myc-DDK expression vector (transmembrane NCAM2 isoform) or empty vector (EV) as a control and then treated with DMSO only, GI254023X (GI; selective ADAM10 inhibitor, 5 μM in DMSO), C3 (β-secretase inhibitor IV, 10 μM in DMSO), GM6001 (GM; broad spectrum of MMPs inhibitor, 2.5 μM in DMSO), and DAPT (γ-secretase inhibitor, 10 μM, in DMSO) for 24 h as indicated. Total DNA concentration was kept constant at 1.5 μg with empty vector. A, representative immunoblot of cell lysates (Lysate) and conditioned media (CM) using anti-Myc (2272), anti-NCAM2 (sc-136328), anti-BACE1 (D10E5), and anti-GAPDH (MAB374) antibodies. Ectopically expressed full-length NCAM2 (NCAM2-FL) in HEK cells undergoes proteolysis with the generation of a C-terminal fragment (NCAM2-CTF) in cell lysates and secreted soluble fragments (sNCAM2) in conditioned media. GI and GM treatments produced a significant reduction of sNCAM2 levels indicating that NCAM2 is cleaved by ADAM10 and MMPs. DAPT treatment results in the accumulation of NCAM2-CTF in cell lysates indicating that NCAM2-CTF is further processed by γ-secretase. C, HEK cells were cotransfected with NCAM2 and BACE1 or EV as control and then treated with solvent only (DMSO), C3, or DAPT for 24 h. The ectopic expression of BACE1 produces BACE1-specific NCAM2-CTF (NCAM2-βCTF) and soluble NCAM2 fragment (sNCAM2β), and their production is completely inhibited by C3 treatment. Note that a different protein ladder was used in this figure compared to Figure 1. B and D, graphs represent densitometric quantification of NCAM2-CTF/NCAM2-FL, NCAM2-FL/GAPDH, NCAM2-CTF/GAPDH ratio in lysates, and secreted soluble NCAM2 (sNCAM2 or sNCAM2β) in CM. One-way ANOVA with Tukey's multiple comparison test was applied. ∗p < 0.05, ∗∗<0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, A and B, n = 3; C and D, n = 4. Full-length versions of the western blots in C are shown in Figure S4A. ns, not significant.
Fig 5: Modified FTO electrode optimization: (A) DPV of rGO-immobilized FTO sensor, where the rGO concentration was decreased from 1, 0.5, 0.25 to 0.1 mg/ml, showed maximum signal at 0.5 mg/ml; (B) DPV of Ab-immobilized FTO/rGO electrode where BACE1 Ab concentration was decreased from 1.5, 1, 0.5 to 0.25 µg, showed optimum signal at 1 μg; (C) DPV of FTO/rGO/BACE1Ab electrode where pH was increased from 6, 6.5, 7, 7.5 to 8, and pH 7.5 gave maximum current output; (D) DPV of FTO/rGO/BACE1Ab electrode where temperature was increased from 4°C, RT, 37°C to 45°C, and room temperature showed the highest current output.
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