Rabbit monoclonal anti nlrp3

Frozen sections (thickness, 10 μm) of the mice brain were blocked with 5% normal goat serum in TBS for 30 min, followed by incubation with primary antibodies overnight at 4°C in TBS containing 5% goat serum and 0.1% Triton. Primary antibodies used included mouse monoclonal anti-Aβ (1:250), rabbit monoclonal anti-NF-κB p65 (1:50, Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-NLRP3 (1:100, Cell Signaling Technology, Danvers, MA, USA). The antibody used for NF-κB in the following experiments was probing the p65 subunit. After washing with TBS, the sections were incubated with anti-mouse IgG (H+L), F(ab')₂ fragment (Alexa Fluor 488 Conjugate) (1:1000, Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit IgG (H+L), and F(ab')₂ fragment (Alexa Fluor 594 Conjugate) (1:1000, Cell Signaling Technology, Danvers, MA, USA) in TBS containing 5% goat serum and 0.1% Triton at room temperature for 1 h. The double immunostaining was analyzed using a laser scanning confocal microscope (A1, Nikon, Shanghai, China).

ox-LDLs were purchased from Yiyuanbiotech (Guangzhou, China). Phorbol-12-myristate-13-acetate (PMA), rapamycin, 3-methyladenine (3-MA), bafilomycin A1 and protease inhibitor cocktails were purchased from MCE (Monmouth Junction, USA). LPS and ATP were purchased from Sigma (St Louis, MO, USA). Lipofectamine™ 2000 was purchased from Invitrogen (Carlsbad, USA). Antibody to GAPDH, antibody to β-actin and HRP-conjugated secondary polyclonal antibodies to mouse and rabbit IgGs were purchased from Elabscience (Wuhan, China). Rabbit monoclonal anti-NLRP3, anti-ASC, anti-SQSTM1/p62, anti-LC3B, antibody to K63-linked polyubiquitin chains, antibody to K48-linked polyubiquitin chains and p62-siRNA, and control siRNA were purchased from Cell Signaling Technology (Boston, USA). Rabbit monoclonal anti-IL-1β, anti-caspase-1 were purchased from Abcam (Cambridge, MA, USA). For the immunoprecipitation, polyclonal rabbit anti-SQSTM1/p62 was purchased from Abcam (Cambridge, MA, USA). Monoclonal mouse anti-SQSTM1/p62, anti-NLRP3, anti-ASC and protein A/G Plus-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AMSH (the JAMM/MPN+ family of K63-specific DUB) was purchased from Boston Biochem (Cambridge, MA, USA).

Tenuigenin (molecular formula: C₃₀H₄₅ClO₆; average molecular weight: 537.14 kDa, chemical structure showed in Fig. 1) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (111572-200702) with a purity of 98.7%. LPS (Escherichia coli 0111:B4, L4391), MPTP (M0896), ATP (A2385), and MSU (U2875) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse IL-1β ELISA Kits were purchased from R&D Systems. ROS-specific fluorescent probe cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Invitrogen (Thermo Fisher Scientific Inc., USA). The antibodies used in this study are the following: rabbit monoclonal anti-NLRP3 (1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-caspase-1 (1:800, Santa Cruz Biotechnology, USA), goat anti-IL-1β (1:800, R&D Systems, Minneapolis, USA), mouse monoclonal anti-β-actin (1:5000, Sigma-Aldrich), mouse monoclonal anti-tyrosine hydroxylase (TH, 1:1000, Sigma-Aldrich), and rabbit polyclonal anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:500, Wako, Osaka, Japan). The reagents obtained from other sources are detailed throughout the following text.Fig. 1

Chemical structure of tenuigenin