Ab181615

Protein was extracted from the platelets or brain homogenates using RIPA buffer supplemented with a protease inhibitor and quantified using a bicinchoninic acid (BCA) (23,227, Thermo, USA) kit. Equal amounts of protein from each sample were separated using 8% SDS-PAGE, transferred onto a nitrocellulose membrane, and incubated overnight with antibodies targeting p-RIP1 (Ser166, 1:1000, 53,286, CST, USA), RIP1 (1:1000, ab202985, Abcam, USA), p-RIP3 (Ser232, 1:1000, ab195117, Abcam, USA), RIP3 (1:1000, ab62344, Abcam, USA), p-AKT (Ser 473, 1:1000, 4060 T, CST, USA), AKT (1:1000, 4691S, CST, USA), Fosb ( 1:1000, ab11959, Abcam, USA), Jun (1:1000, ab40766, Abcam, USA), Jund (1:1000, ab181615, Abcam, USA), Fos (1:1000, ab222699, Abcam, USA), Junb (1:1000, 128,878, Abcam, USA), and β-actin (1:5000, A5441, Sigma) in Tris buffered saline containing 0.2% Tween-20 (TBST) and 5% nonfat dry milk at 4 °C. After washing with TBST, the membranes were incubated with 1 μg/ml goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye 800CW (Licor Odyssey, USA). The positive bands were detected using the Odyssey infrared imaging system (LI-COR Biosciences, USA), and signal intensity was quantified using ImageJ software and normalized to that of β-actin.