Sc 9149

ESCs and CSCs were cultured on coverslips, and the medium was replaced with SFM containing MCC950 (100 μM). After incubation for 16 h, the cells were fixed in methanol for 2 min at room temperature and permeabilized with 0.5% Triton X-100 for 1 min. After blocking with 1% bovine serum albumin for 1 h at room temperature, the cells were incubated with primary antibodies against NLRP3 [19771–1-AP, 1:200, Proteintech, Chicago, IL, USA], IL-1β [sc-32294, 1:100, Santa Cruz Biotechnology, Heidelberg, Germany], CD10 [sc-9149, 1:50, Santa Cruz Biotechnology], vimentin [sc-6260, 1:50, Santa Cruz Biotechnology], and fibronectin [ab2413, 1:200, Abcam, Cambridge, UK] for 2 h at room temperature. The cells were then incubated with goat anti-rabbit (Alexa Fluor® 568, 1:500, Thermo Fisher Scientific, for anti-NLRP3 antibody) and goat anti-mouse (Alexa Fluor® 488, 1:500, Thermo Fisher Scientific, for anti-IL-1β antibody) secondary antibodies for 1 h at room temperature. Nuclear staining was carried out using 4',6-diamidino-2-phenylindole (4083, 1:1000, Cell Signaling Technology). Visualization was performed using a confocal laser-scanning microscope (BZ9000, Keyence, Osaka, Japan).

Brain homogenates were prepared as we described previously [16] (link), [17] (link). Commercial ELISA kits (Invitrogen) were used to measure Aβ40 and Aβ42 levels in carbonate soluble and insoluble (guanidine soluble) fractions according to the manufacturer's protocol. For immunoblot analysis, aliquots of brain homogenate were separated by SDS-PAGE and blotted to nitrocellulose or polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with specific primary antibodies against the carboxyl terminus of APP (CT695; Invitrogen), the soluble amino-terminal fragments of APP (sAPPα and sAPPβ) (2B3 (11088B) and 6A1 (10321B), Clontech), mouse apolipoprotein E (apoE) (sc-6384, Santa Cruz Biotechnology), insulin-degrading enzyme (IDE) (PC730, EMD Biosciences/Millipore), neprilysin (sc-9149, Santa Cruz Biotechnology), ionized calcium-binding adaptor molecule 1 (IBA1) (016-20001, Wako), and nitric oxide synthase 2 (NOS2) (sc-7271, Santa Cruz Biotechnology) followed by HRP-conjugated secondary antibodies. Signal was detected by the ECL plus Western Blotting System (GE Healthcare) and quantified by ImageJ software. For a loading control when appropriate, the blots were stripped and reprobed with mouse anti-actin antibody (MAB1501R, Millipore) or mouse anti-tubulin antibody (T5168; Sigma).