Atpase assay kit

Whole-cell lysates were prepared from the primary cortical astrocytes as described above for the ATP quantification. Contamination with inorganic phosphate (Pi) was removed via incubation of the lysate with Pi Bind resin (Innova Biosciences) for 2 h at +4°C. ATPase activity was quantified in aliquots of 10 μg of protein using ATPase assay kit (Innova Biosciences) according to the manufacturer’s instructions. The amount of Pi released was quantified colorimetrically at 630 nm using Infinite M1000 Pro multi-mode microplate reader (Tecan). Pi standard curve was established in each experiment.

The malachite green assay (ATPase assay kit, Innova Biosciences) was used to evaluate the ATPase activity of NLRP1. The assay was performed by incubating purified recombinant NLRP1 constructs with ATP and measuring the concentration of free phosphate released in solution upon ATP hydrolysis. The protein concentration used in the assay was 0.1 mg/mL in 25 mM Hepes pH 7.0, 150 mM NaCl, 1mM MgCl₂ and 1 mM DTT while the initial ATP concentration was 1 mM. The assay was performed at room temperature for 1 hour and the concentration of free phosphate in solution was monitored before adding ATP and after 5, 20, 40 and 60 minutes of incubation. Free phosphate in solution was detected by adding green malachite reagent and by measuring the UV absorption at 620 nm. The assay was also repeated in presence of 0.1 mg/mL MDP. A positive control experiment was also performed using a solution of DnaK at 0.1 mg/mL in presence of 1 mM ATP.

The ATPase activity in the SR vesicles was determined using an ATPase assay kit (Innova Biosciences, Cambridge, UK) according to the manufacturer’s instructions with minor modifications. Briefly, SR vesicles (0.1 μg) in a total volume of 100 μl were incubated for 10 min in the presence of various concentrations of cADPR (0–40 μM) with or without a 10 min pretreatment with various inhibitors. The mixture was incubated for 1 min at 37 °C in substrate buffer (0.5 M Tris-HCl, 0.1 M MgCl₂, 10 mM purified ATP, 5 μM calcein-AM, 5 mM sodium azide, and 1 mM ouabain, pH 7.4) with or without Na-orthovanadate (2–20 μM). After incubation, the reaction was stopped by the addition of 50 μl of Gold Mix from the ATPase assay kit. Two minutes later, 20 μl of stabilizer from the ATPase assay kit was added, and the solution was incubated for 20 min at 37 °C in the dark. The enzyme activity was calculated by measuring the Pi-dye complex released via ATP hydrolysis using an ELISA plate reader at 635 nm (Molecular Devices, Sunnyvale, CA, USA).

ATP hydrolysis was measured using a colorimetric ATPase Assay Kit (Innova Biosciences, Cambridge, UK) following the manufacturer's instructions. Briefly, 0.2 μm myosin S1, 1 mm fresh ATP, and 0.4 mg·mL⁻¹ actin filaments were mixed with 2 μm of UNC‐45B, central domain or UCS domain in 10 mm Tris, 50 mm KCl, 10 mm MgCl₂, 0.2 mm CaCl₂, and 1 mm DTT. Each condition was assayed using 100 μL aliquots of the reaction mix at each time point, promptly quenched, and then the absorbance was measured at 650 nm (using a Molecular Devices VersaMax Tunable Microplate reader, Molecular Devices, Silicon Valley, CA, USA). These reactions were carried out at 25 °C.

ATP hydrolysis was measured using an ATPase Assay Kit (Innova Biosciences) following the manufacturer's instructions. 1 μg Hsp70 and Hsc70 recombinant proteins used in the in vitro acetylation assay with or without 1 μg ARD1 recombinant were incubated with 1 μg Hsp40 recombinant (ATGen) in a reaction buffer consisting of 50 mM Tris (pH 7.5), 2.5 mM MgCl₂ and 0.5 mM ATP at room temperature. For cells, GFP-Hsp70 precipitated from 1 mg cell extracts was used for reaction. After 60 min, PiColorLock Gold reagent and Accelerator were added to the solution. Stabilizer was added 2 min later, and the resulting green colour was allowed to develop for 30 min at room temperature. Absorbance was measured at 595 nm.