Fig. 2. ATP-hydrolysis activity of NLRP3 determined by reverse-phase HPLC.

a Separation of adenosine nucleotides by HPLC on a RP-18 column. Nucleotides elute at about 3 mL (AMP), 4.5 mL (ADP) and 8.5 mL (ATP). b MBP-NLRP3 peak 1 or peak 2 (3 µM) were incubated at 25 °C for 68 min in the presence of 5 mM MgCl₂ and 100 µM ATP. The amount of AMP, ADP and ATP was determined in 10 min intervals using RP-HPLC. The eluted peaks were evaluated by integration and the total integral adjusted to 100%. The relative ATP concentration is shown on the y-axis and the time on the x-axis (min). Shown is one representative measurement of n > 5 biologically independent experiments. c The hydrolysis activity of MBP-NLRP3 peak 1 depends on MgCl₂. MBP-NLRP3 peak 1 (3 µM) was incubated at 25 °C for 68 min in the presence of 100 µM ATP, but in the absence of MgCl₂. Shown is one representative experiment of n = 2 independent experiments. d ATP is hydrolyzed by NLRP3 peak 1 to ADP. MBP-NLRP3 peak 1 at 3 µM concentration was incubated at 25 °C for 68 min in the presence of 5 mM MgCl₂ and 100 µM ATP. The relative amount of nucleotide is shown for ATP and ADP. Shown is one representative measurement of n > 5 biologically independent experiments.