3.4. In Vitro Measurement of NLRP3 ATPase Activity

Human NLRP3 ({"type":"entrez-nucleotide","attrs":{"text":"NM_004895.4","term_id":"208879434","term_text":"NM_004895.4"}}NM_004895.4) was cloned into the pAcGFP-N1 vector (ClonTech, Product# 632469) as previously described [46]. For heterologous expression, the NLRP3-GFP plasmid was transfected into HEK293T cells with PolyJet (SignaGen Laboratories, Catalog #SL100688) according to the manufacturer’s instructions. Approximately 12 h after transfection, cell lysates were prepared in GFP-Trap Lysis Buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA with 0.5% (v/v) NP-40). Immobilised GFP-Trap Sepharose resin was produced according to the reported procedure [49]. The GFP-Trap clone was a gift from Dr. Laura Trinkle-Mulcahy (University of Ottawa). The capture of NLRP3-GFP protein from HEK293 lysates with GFP-Trap was performed according to the Chromotek GFP-Trap protocol with some modifications. Cleared lysates were diluted 1:2 with GFP-Trap Wash Buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA) and incubated with GFP-Trap beads with end-over-end mixing for 1.5 h at 4 °C. The bead slurry was transferred to a small gravity column, and the GFP-Trap resin was washed sequentially with High-Salt Wash Buffer (10 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.5 mM EDTA) and GFP-Trap Wash Buffer. For measurement of ATPase enzymatic activity, the GFP-Trap beads were subjected to a final wash of 4 × 2 mL with ADP-Glo Reaction Buffer A (40 mM Tris-HCl pH 7.5, 20 mM MgCl₂ and 0.1 mg/mL BSA).

The ATPase activity of the NLRP3-GFP protein was determined “on-bead” following GFP-Trap capture using the luminescent ADP-Glo Kinase Assay Kit (Promega). The GFP-Trap beads were resuspended in 375 μL of ADP-Glo Reaction Buffer A, and 20 μL aliquots of the bead slurry were distributed to wells of a 96-well plate. All ATPase reactions were performed in Reaction Buffer A (40 mM Tris-HCl pH 7.5, 20 mM MgCl₂ and 0.1 mg/mL BSA) supplemented with cOmplete™, EDTA-free Protease Inhibitor Cocktail Tablets (Millipore Sigma Cat# 11836170001). Reactions were incubated with NLRP3 inhibitor or vehicle (DMSO) and then started by addition of ATP (to give × mM final concentration). The reactions were carried out in white, flat-bottomed 96-well plates (Corning^® 96 Well Solid Polystyrene Microplate, Sigma Aldrich, Cat# CLS3917) with gentle-vibrational mixing at 37 °C for 10 min. After the reaction incubation period, the assay was terminated by addition of ADP-Glo Reagent (25 μL) and incubation on a shaker at room temperature for 40 min. Subsequently, Kinase Detection Reagent (50 μL) was added to each well and incubated with shaking at room temperature for 60 min before luminescence was recorded with a Spectra Max M2 plate reader. A standard curve for ATP to ADP conversion was included on each 96-well plate to convert Relative Luminescence Units (RLU) into the amount of ADP present. To account for background signal, RLUs of blank wells containing no ATP were subtracted from the reaction well values. All assays were determined to be linear with respect to time and NLRP3-GFP content.