Anti glua2

Samples were denatured in Laemmli buffer/β-mercaptoethanol and heat (100 °C) for 10 min and resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were transferred to a nitrocellulose membrane (Bio-Rad, Berkeley, CA, USA) for subsequent detection, using immunoblotting. Blots were blocked for 1 h at room temperature with 5% (w/v) nonfat dry milk in tris buffered saline (0.1% v/v Tween-20, pH 7.5 (TBS-T)). Blots were then incubated with the following primary antibodies, overnight, at 4 °C: anti-PICK1 (1:1000, rabbit IgG, Epitomics, Burlingame, CA, USA); anti-GluA2 (1:1000, rabbit IgG, AbCam, Cambridge, MA, USA); anti GluA1 (1:1000, rabbit IgG, Epitomics, Burlingame, CA, USA); anti-β-Actin (1:5000, mouse IgG, Calbiochem, San Diego, CA, USA). Then they were washed 3 times with TBS-T and incubated with rabbit or mouse horseradish peroxidase secondary antibody (1:2000, Jackson ImmunoResearch, West Grove, PA, USA), for 2 h, at room temperature. Blots were then washed 3 times in TBS-T and then incubated with chemiluminescence substrate (Thermo, Rockland, IL, USA). Immunoreactive bands were visualized and captured with a Fuji Imaging System. Blots were stripped and re-probed up to 2 times, using Restore Plus Western Blotting Stripping Buffer (Thermo, Rockland, IL, USA).

Anti-NLRP3 (#ab91413), anti-caspase-1 (#ab1872), anti-IL-1β(#ab9722), anti-GluA1 (#ab31232), anti-GluA2 (#ab133477), anti-sodium potassium ATPase (#ab76020) and anti-VCP/p97 (#ab11433) antibodies were purchased from Abcam. Anti-β-actin antibody (#A5441) was obtained from Sigma-Aldrich. Anti-PSD-95 antibody (#MAB1598) was obtained from Millipore.
The Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (#SM-005) was purchased from Invent Biotechnologies, Inc. Roche Applied Science provided Complete Protease Inhibitor Cocktail Tablets (#04693116001). The Pierce™ BCA Protein Assay Kit (#23225) was purchased from Thermo Scientific. Protein A/G Magnetic Beads (#B23202) was purchased from Bimake. AC-YVAD-CMK peptide (#SML0429) was purchased from Sigma-Aldrich. For the in vitro experiments, cultured primary neurons were pretreated with AC-YVAD-CMK (5 µM) 1 h before oxygen-glucose deprivation (OGD). For the in vivo experiments, AC-YVAD-CMK (1 mg/kg, i.p.) was administered 1 h before HIBD surgery and then daily for 7 days.