Ab7149

Western blot was completed as previously described by us [5 (link),21 (link),22 (link)]. In brief, the soleus muscle was homogenized using radioimmunoprecipictation assay cell lysis buffer and the supernatant was used. Using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA), protein concentration was estimated and 50 µg of protein was run onto a 10% or 15% SDS-PAGE gel (150 V for 1 h). Using a semi-dry transfer machine (Bio-Rad), gels were transblotted onto polyvinylidene difluoride membranes (Bio-Rad) and were subsequently blocked with 5% skim milk. Next, the membranes were incubates with primary antibodies overnight at 4 °C for inflammasome markers including TLR4 (1:1000, Cat# ab13556, 90 kDa; Abcam) and NLRP3 (1:1000, Cat# ab214185, 90 kDa; Abcam). Pyroptosis markers were detected using primary antibodies for IL-1β (1:1000, Cat# ab2105, 31 kDa; Abcam), and IL-18 (1:1000, Cat# ab7149, 22 kDa; Abcam). Inflammation was detected by primary antibody for TNF-α at a 1:500 dilution (Cat# ab6671, 17–22 kDa; Abcam). Finally, primary antibody for β-actin was used as a loading control at a 1:1000 dilution (Cat# ab16039, 45 kDa; Cell Signaling Technology, Danvers, MA, USA). Membranes were exposed to enhanced chemiluminescence substrate (ThermoFisher Scientific). Densitometric analysis was performed using NIH ImageJ software.

Human embryonic kidney 293 (HEK293) cells were transiently transfected with NMDAR subunit genes (NR1/NR2A; DsRed2-labeled) as previously described [14 (link)]. Twenty-four hours later, cells were fixed on coverslips with acetone and incubated overnight at 4 °C with the purified ABs from patients or CSF from immunized mice (starting at a 1:1 ratio) in PBS containing 5 % bovine serum albumin (BSA). After washing with PBS, the cells were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-human IgG (ab7149, Abcam) or FITC-conjugated anti-mouse IgG (ab6785, Abcam) and observed under a fluorescence microscope (BX51, Olympus, Japan).