Supplementary Material for: RIP3 augments neuroinflammation by facilitating neutrophil infiltration during an ischemic stroke.

Introduction: Neutrophil infiltration is responsible for the neuroinflammation during an ischemic stroke. Here, we explored the role of receptor-interacting protein kinase 3 (RIP3) in neutrophil infiltration during an ischemic stroke.
Methods: The rat middle cerebral artery occlusion (MCAO) model was utilized to identify pivotal proteins involved in neutrophil infiltration during an ischemic stroke. Neutrophils were isolated from the peripheral blood of mice, and a co-immunoprecipitation (co-IP) assay was performed to identify the proteins that interact with RIP3.
Results: The rat MCAO model was successfully established. Myeloperoxidase (MPO) was significantly up-regulated in the MCAO group, indicating the presence of neutrophil infiltration. RIP3 protein level exhibited a similar trend to MPO protein level, suggesting that neuroinflammation might be partly activated by RIP3 through the promotion of neutrophil infiltration. Co-immunoprecipitation (co-IP) and mass spectrometry (MS) analyses suggested that RIP3 facilitated neutrophil infiltration partly by affecting protein kinases (Rock1 and Prkaca) downstream of RIP3, and the interaction between RIP3 and Rock1 or Prkaca was validated by IF and co-IP assays.
Conclusion: In this study, it was observed that RIP3 affects neutrophil infiltration, a critical phenomenon associated with neuronal injury during ischemic stroke, partly by the modulation of downstream proteins such as Rock1 and Prkaca.