Af2767

The following primary antibodies were used: goat polyclonal anti-adiponectin (AF1119, R&D Systems); goat polyclonal anti-T-cadherin (AF3264, R&D Systems); rabbit monoclonal anti-α-tubulin (11H10, Cell Signaling Technology); sheep polyclonal anti-human MFG-E8 (AF2767, R&D Systems); mouse monoclonal anti-human CD63 (H5C6, BD Biosciences); rabbit monoclonal anti-Tsg101 (ab125011, R&D Systems); rabbit polyclonal anti-syntenin (ab19903, Abcam); and mouse monoclonal anti-ALIX (3A9, Santa Cruz Biotechnology). The following secondary antibodies were used: horseradish-peroxidase-conjugated (HRP-conjugated) rabbit anti-sheep immunoglobulin G (IgG) (Invitrogen); HRP-conjugated donkey anti-goat IgG (R&D systems); and HRP-conjugated sheep anti-mouse IgG antibodies and donkey anti-rabbit IgG antibody (GE Healthcare).

Equal protein concentrations of protein-extracted synaptoneurosome samples were applied on nitrocellulose membrane using a vacuum dot blotter (CSL-D48, Cleaver Scientific). Membranes were air-dried and blocked with Odyssey blocking buffer (LI-COR, 927–40000) for 30 min, followed by incubation with an anti-MFG-E8 antibody (sheep, R&D Systems AF2767, 1:500) and rabbit anti-goat HRP-tagged secondary antibody (Abcam ab6741, 1:5000). Membranes were processed with SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific, 34075) for 5 min and imaged on an LI-COR Scanner under chemiluminescence settings. Total protein was measured with Revert 700 (LI-COR, 926–11021). For each blot, the same size box was used to ensure all samples are measured equally. Each sample was normalized to its corresponding value of total protein. Mouse hippocampus was used as a negative control and human milk was used as a positive control.