Extended Data Fig. 5. Validation of BACE1 and APP antibodies with KO materials.
(a) Confocal images of primary OLs harvested from Control;AD and OL-Bace1cKO;AD mice labeled with PLP (yellow), APP (blue), and 3D5 for BACE1 (white). The punctate 3D5 labeling is equally present in Control;AD and OL-Bace1cKO;AD, suggesting an unspecific BACE1 staining. Experiment was repeated twice in separate in vitro cultures. (b) Confocal images of primary OLs harvested from Control;AD and OL-Bace1cKO;AD mice labeled with PLP (yellow), APP (blue), and ab183612 for BACE1 (white). No BACE1 reactivity could be detected. Experiment was repeated twice in separate in vitro cultures. (c) Fluorescence microscopy images of mossy fibers of 9-month-old WT (left) and constitutive BACE1 KO (right) animals labeled with 3D5 (top) and ab183612 (bottom) for BACE1 (white). Loss of mossy fiber staining was only detected via 3D5 labeling. Immunolabeling was performed once on brain slices from different mice (n = 2 per group). (d) Confocal images of 9-month-old WT (top) and constitutive BACE1 KO (bottom) cortices labeled with DAPI (gray) and 3D5 for BACE1 (blue). Note the absence of any intracellular staining of 3D5 in both WT and BACE1 KO animals, hinting at the inability of the 3D5 antibody to detect BACE1 in cell somas. Immunolabeling was performed once on brain slices from different mice (n = 2 per group). (e) Immunoblot representative images of BACE1 and the loading control, actin, on sorted OLs from 1-month-old Control;AD and OL-Bace1cKO;AD (n = 1 per group), showing a proof of concept ablation of BACE1 in a cell-type-specific manner. (f) Fluorescence microscopy images of piriform cortices of 3-month-old WT (left) and constitutive APP KO (right) animals labeled with CAII (yellow) and Y188 for APP (blue) for BACE1 (white). APP reactivity, including the APP labeling in OL soma (insets), is abolished in the KO tissue. Immunolabeling was performed once on brain slices from different mice (n = 2 per group). (g-h) Cell-type-specific deletion of Bace1 alters APP processing. (g) Immunoblots and total protein content of microdissected cortical and WM tissues from 6-month-old male mice targeting key amyloidogenic proteins in lysates. (h) Immunoblot quantification showing APP processing in WT, control, ExN-Bace1cKO;AD and OL-Bace1cKO;AD (n = 4–6 per group) lysates. Top–cortical, bottom–WM. All immunoblots were normalized to WT relative protein amount except β-CTFs which were normalized to control AD relative protein amount. Data was statistically analyzed via one-way ANOVA was performed with Tukey multiple comparison tests (P values indicated in graphs with significance highlighted in bold). Bars represent means with SEM and individual data points displayed.