Fig. 2.

ATX activity cannot be measured by the FS-3 assay in biological samples if lysophospholipids are present. A: Recombinant ATX (75, 100, and 125 ng/ml) was incubated with or without 10 nM of ONO-8430506 using 5 μM of FS-3. ONO-8430506 suppressed >97% of the total activity measured in the assay. B: The same experiment in (A) was conducted using 0.25–10 μl of plasma from three different mice (final assay volume of 100 μl). ONO-8430506 could not suppress the measured activity and is therefore not from ATX activity. C: LPC potently inhibits ATX activity (ATX at 100 ng/ml concentration) when incubated with FS-3. D: Removing lipids from FBS reveals a component of the measured total activity that can be suppressed by ONO-8430506. These results are obtained from the subtraction of the total measured activity from the activity remaining after adding 10 nM ONO-8430506 (see supplementary Fig. 1). All results are mean ± SEM from three independent experiments.