Af 501 na

To detect precursor and mature IL-1β expression, LPS (500 ng/mL for 3 h)-primed rat microglial cells were treated with compounds (10 μM for 30 min) and then treated with ATP (2 mM for 30 min, co-treatment with each compound). After stimulation, cells (lysates) and culture medium (supernatants) were isolated, and each sample lysed in RIPA buffer (Nacalai Tesque, Inc., Kyoto, Japan). The samples were subjected to a 10% polyacrylamide gel, and the proteins were transferred electrophoretically to PVDF membranes (GE Healthcare, Tokyo, Japan). After blocking with blocking buffer (Nacalai Tesque, Inc., Kyoto, Japan), membranes were incubated with anti-IL-1β, rat, goat-poly (1:1000; AF-501-NA, R&D systems) and β-actin (1:5000; Sigma-Aldlich, St. Louis, MO, USA). The antibodies were detected using an HRP-conjugated secondary antibody (1:1000, GE Healthcare, Tokyo, Japan) and visualized using an ECL system (GE Healthcare, Tokyo, Japan).

Rat hippocampal tissue was homogenized in RIPA buffer (Thermo Scientific, Waltham, MA, USA), which contained protease inhibitors (Beyotime, Jiangsu, China). The protein samples were electrophoresed on 12% Tris-glycine SDS-PAGE gels. Then the gels were transferred to PVDF membranes (0.2 or 0.45 μm), followed by blotting with antibodies against Iba-1 (1:1000, Ab5076, Abcam), NLRP3 (1:1000, #768319, R&D), Caspase-1 p20 (detects endogenous levels of pro-caspase-1 and the caspase-1 p20 subunit) (1:1000, A27351509, AdipoGen), ASC (1:200, Sc-22514-R, Santa Cruz), IL-1β (1:1000, AF-501-NA, R&D), GAPDH (1:20,000, 60004–1–1g, Proteintech), and β-actin (1:20,000, HRP-60008, Proteintech). Primary antibody incubation was performed at 4 °C overnight. Afterward, the membranes were incubated with the secondary antibody (1:10,000) at room temperature for 2 h. The signal was captured on an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Buckinghamshire, UK) (n = 4/group). The protein expression was quantified by analyzing band levels with ImageJ software (NIH, Bethesda, MD, USA).