Protein Extraction for Western Blot Analysis

Protein extraction for WB analysis was performed as reported by De Vreese and colleagues (2019) [38 (link)], using frozen brain tissue from bottlenose dolphin, fin whale, Cuvier’s beaked whale, striped dolphin and sperm whale specimens. Overnight incubation at 4°C followed, using polyclonal rabbit Apaf-1 (Enzo, #ADI-905-179-100, 1:1000), anti-DGK-ζ (MyBiosourse, #MBS2026991, 1:500), anti-Bcl-2 (Abcam, #ab196495, 1:1000), anti-MDA (Abcam, #ab6463, 1:1000) and anti-iNOS (Abcam, #ab15323, 1:250); a monoclonal recombinant rabbit anti-Aβ (ThermoFisher Scientific, #700254, 1:500); and monoclonal mouse anti-NF200 (Sigma-Aldrich, #N0142), anti-GAD⁶⁷ (Sigma-Aldrich, #MAB5406, 1:2000), anti-GAP43 (Sigma-Aldrich, #MAB347, 1:1000), anti-TNFα (Santa Cruz, #sc-52746, 1:200), anti-QKI-7 (Antibodies-online, #ABIN1304925, 1:1000) and anti-synaptophysin (Dako; #M7315, 1:1000). After several washes in TBS-T, the membrane was incubated with an anti-rabbit peroxidase-conjugated secondary antibody (ThermoFisher Scientific, #32260) for 1 hour at room temperature. In order to visualize immunoreactive bands, a chemiluminescent detection kit (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific) and the iBright machine (ThermoFisher Scientific) were employed.