*4.6. Quantification of PPi Levels in Medium Samples*

We have previously found that HEK293 cells endogenously express ENPP1 [10] and that PPi accumulation in the medium can be used as a robust secondary assay to determine the amount of ATP released by cells into the culture medium. Using two independent assays to follow ATP efflux provides robust data on the activity of the studied rAbcc6 mutants. PPi concentrations in cell culture medium samples were determined as described previously [14,19]. Briefly, PPi was quantitatively converted into ATP by incubating samples and standards in an assay containing 50 mM HEPES pH 7.4, 80 µM MgCl2, 32 mU/mL ATP sulfurylase (New England Biolabs, Ipswich, MA, USA), and 8 µM adenosine 5'-phosphosulfate (Santa Cruz Biotechnology, Dallas, TX, USA) at 30 ◦C for 30 min and the reaction was terminated by incubating the samples at 90 ◦C for 10 min. ATP levels were then determined in the reaction mix by bioluminescence by adding BacTiterGlo reagent (Promega, Madison, WI, USA) in a 1:1 ratio in a total volume of 40 µL. PPi concentrations in medium samples were then calculated by interpolation from a standard curve. The values were adjusted by subtracting background provided by controls in which ATP sulfurylase was omitted.
