Ab19026

Immunoblot and serial fractionation were performed as previously described^{46 (link)} with the following antibodies: rabbit monoclonal anti-BACE1 (1:1000; D10E5; Cell signaling technology); mouse monoclonal anti-APP (and APP CTFs) antibody (1:5000; C1/6.1; BioLegend); goat polyclonal anti-N-terminal CHL1 antibody (for CHL1-FL and CHL1-NTF) (1:1000; AF2147; R&D Systems); mouse monoclonal anti-GAPDH (1:10,000; MAP374; Millipore); mouse monoclonal anti-β-tubulin (1:10,000; JDR.3BR; Sigma); mouse monoclonal anti-calnexin (1:2000; 610523; BD biosciences); rabbit polyclonal anti-ADAM10 (1:1000;AB19026; Millipore); rabbit polyclonal anti-PS1 AB14 (1:1000)^{47 (link)} and rat monoclonal anti-SEZ6 (1:250)^{25 (link)}, rat monoclonal anti-APPsβ (1:40)^{48 (link)} and HRP-conjugated secondary antibodies visualized by ECL (GE Healthcare). Chemiluminescent signal was captured on an LAS4000 Fuji Imager. Densitometry analysis was performed using Quantity One software (Bio-Rad Laboratories).

To analyze the endogenous localization of ADAM10 (1:100, AB19026, Millipore) and CX3CL1 (1:100, ab25088, Abcam), heart tissue sections (mid-ventricular short axis sections, 4% PFA fixed, paraffin embedded, 3 µm) and indicated cells were stained with specific antibodies and counterstained with CD31 (1:100, 553370, BD Biosciences), CD45 (1:100, 555480, BD Biosciences), cTnI (1:100, MAB3150, Millipore), cTnT (1:100, ab45932, Abcam) or Vimentin (1:100, 550513, BD Biosciences) where indicated. Alexa Fluor 488-coupled goat anti-rabbit (1:200, A27034, ThermoFisher Scientific), Alexa Fluor 546-couppled goat anti-mouse (1:200, A-11003, ThermoFisher Scientific) or CF 594-coupled goat anti-rat (1:200, SAB4600323, Sigma-Aldrich) and ProLong Diamant Antifade Mountant with DAPI (P36962, ThermoFisher Scientific) were used as secondary antibodies and for mounting, respectively. Image acquisition was performed using a Keyence BZ-X710 All-in-One Fluorescence Microscope (Keyence) with the BZ-X Viewer v1.03.00.05 software (Keyence). The cellular distribution of ADAM10 and CX3CL1 was assessed using CellProfiler 4⁴⁶ . Cardiac-TnT, CD31, CD45 and Vimentin positive cells were analyzed for ADAM10 expression. For each mouse at least 1000 cells/staining were analyzed. Cardiomyocyte cross-sectional diameter was determined using Fiji. One hundred cells/mouse were analyzed.

Pooled samples, soluble and insoluble fractions were prepared in SDS loading buffer (4X Roti-Load, Carl Roth, Germany) and boiled at 95 °C. Protein extracts were separated by SDS-PAGE, transferred onto nitrocellulose membranes (Amersham Hybond ECL), and then blocked in 5% (w/v) milk powder in TBST. Soluble fractions were used to detect total soluble N-APP (sAPP), using an anti-N-terminal APP antibody (22C11, MAB348, Millipore). Moreover, the following antibodies were used for detection in insoluble fractions: anti-CT15 1:10,000 (APP and CTFs) [33 (link)], anti-PSEN1 (7H8, C-terminal), anti-Nicastrin (N1660, Sigma), anti-ADAM10 (Ab19026, Millipore, Massachusetts, USA), anti-Notch1 (ab27526, Abcam), anti-Notch intracellular domain (NICD, New England Bioscence #2421S), and anti-mep1b (AF2895, R&D Systems, Minneapolis, USA).