Ab195117

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.

IVC tissues containing thrombus were fixed on slides and rinsed with PBS. The slides were blocked in 5% BSA in PBS for 1 h and treated with the primary antibodies at 4°C overnight, namely, rabbit anti-MLKL (p-S345) antibody (1 : 1000; #ab196436; Abcam, Cambridge, United Kingdom), rabbit anti-RIP3 (p-S232) antibody (1 : 50; #ab195117; Abcam, Cambridge, United Kingdom), and rabbit anti-IL-17B antibody (1 : 100; #ab79056; Abcam, Cambridge, United Kingdom). The slides were incubated at 37°C for 40 min with the secondary antibody (goat anti-rabbit IgG antibody, Abcam, Cambridge, United Kingdom) after washing with PBS three times and stained with hematoxylin for 5 min. The images were revealed using HRP-DAB and observed using a Lab.A1 microscope at 200x (Carl Zeiss GmbH, Oberkochen, Germany). The positive expression of the protein was observed by an optical microscope and showed brownish-yellow particles. Five high-power fields were randomly selected for observation using the double-blind method. The Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the positive rate.

Western blotting experiment was described previously (Zhao et al., 2019 (link)), and the detection of RIPK3 or MLKL oligomers was performed according to the method described by Wang et al. (2019) (link). The following antibodies were used for the experiments: anti-FADD (1:1000, 610399) and anti-RIPK1 (1:3000, 610458) (BD Transduction Laboratories, San Jose, CA); anti-phospho-RIPK3 (1:1000, ab195117), anti-MLKL (1:1000, ab172868), anti-phospho-MLKL (1:1000, ab196436) (Abcam, Cambridge, MA, United States); anti-RIPK1 (1:3000, 3493), anti-RIPK3 (1:3000, 15828), anti-Myc tag (1:2000, 2276), anti-HA tag (1:2000, 3724), anti-Flag tag (1:2000, 14793), anti-TRAF2 (1:1000, 4724), anti-phospho-RIPK1 (1:1500, 31122) and anti-phospho-RIPK3 (1:1000, 57220) [Cell Signaling Technology (CST), Beverly, MA]; anti-TRADD (1:1000, ABP52634) and anti-GAPDH (1:1500, ABP52783) (Abbkine, Redlands, CA, United States); anti-β-actin (1:3000, A5441) (Sigma-Aldrich). All western blot assays were performed three times, and the representative results are shown.