Mature il 1β

Protein samples obtained from BMDM cells were separated by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Burlington, MA, USA). These membranes were blocked with 5% skim milk and incubated overnight with primary antibodies against LPA₅ (1:1000, LifeSpan BioScience, Seattle, WA, USA), NLRP3 (1:1000), procaspase 1 (1:1000, Abcam), caspase-1 (1:1000, AdipoGen Life Sciences), pro IL-1β (1:1000, Cell Signaling Technology, Danvers, MA, USA), mature IL-1β (1:1000, Abcam), and β-actin (1:10,000, Bethyl Laboratories, Montgomery, TX, USA) followed by incubation with HRP-conjugated secondary antibodies (1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA). Protein bands were visualized using an enhanced chemiluminescence detection kit (Donginbiotech Co., Seoul, South Korea). Image J software was used to quantify target protein bands.

Proteins were extracted from either the ipsilateral brain hemisphere at one day after ischemic challenge or cultured BMDMs using a neuronal protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). These membranes were blocked with 5% skim milk, incubated overnight with primary antibodies against NLRP3 (1:1000), pro-caspase 1 (1:1000, Abcam, Cambridge, UK), cleaved caspase-1 (1:1000, AdipoGen Life Sciences), pro-IL-1β (1:1000, Cell Signaling Technology, Danvers, MA, USA), mature IL-1β (1:1000, Abcam), phospho-NF-κB p65 (1:1000, Cell Signaling Technology), and β-actin (1:10000, Bethyl Laboratories, Montgomery, TX, USA). They were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000, Santa Cruz Biotechnology). Protein bands were detected using an enhanced chemiluminescence detection kit (Donginbiotech Co., Seoul, South Korea). Expression levels of target protein bands were quantified using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA).