Cleaved caspase 1

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).

The cell lines were lysed with lysis buffer (20 mM Tris-HCl, pH 8; 150 mM NaCl; 1% Triton X-100; 10 mM EDTA; 10% glycerol; and 1 mM ZnAc). The proteins were separated via SDS‒polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Amersham Biosciences) and probed with the antibodies against the following proteins: Fibronectin (Merck, Sigma‒Aldrich, 1:1,000), Vimentin (Cell Signaling, 1:1,000), ZEB1 (Atlas Laboratories, 1:1,000), Cleaved Caspase-3 (Cell Signaling, 1:1000), Cleaved Caspase-1 (p20 fragment, AdipoGen, 1:1,000), CD63 (Thermo Scientific, 1:500), and E-Cadherin (mouse polyclonal, Cell Signaling Technology, 1:1,000). For normalization, we used an antibody against β-Tubulin (Elabioscience, 1:2000) or Vinculin (Cell Signaling Technology, 1:10,000). The secondary antibodies, namely, goat anti-rabbit (GeneTex) or anti-mouse HRP (ImmunoReagents), were diluted 1:10,000. The signals were visualized by chemiluminescence using an ECL substrate (Advansta) or by additional sensitive chemiluminescence using a LiteAblot Turbo (EuroClone) following the manufacturer’s instructions.

Cells were lysed using RIPA buffer supplemented with aprotinin (Sigma), and Halt Protease Inhibitor Cocktail (100X) (Thermo Scientific). Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad, Mississauga, ON). Primary antibodies used in this study include caspase-1 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-1 (AdipoGen Life Science, San Diego, CA), caspase-3 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-3 (Cell signaling technology, Danvers, MA), HMGB1 (Cell signaling technology, Danvers, MA), MLKL (Abcam, Boston, MA), phosphorylated MLKL (Abcam, Boston, MA), CRT (Abcam, Boston, MA), and GAPDH (Sant Cruz Biotechnology, Dallas, TX). The expression levels Relative expression of proteins were visualized using the corresponding secondary HRP-conjugated antibodies and Amersham ECL select western blotting detection reagent, as described previously (46 (link)).