Characterizing ENPP1 Variants and Activity

The full-length WT cDNA and mutant human ENPP1 cDNA entailing each of the three variants, c.656G>A, c.1530G>C, and c.876_880delTAAAG, were cloned into a pcDNA3.1(+) vector (GenScript). HEK293 and Cercopithecus aethiops kidney (COS7) cells were transfected using jetPEI (Illkirch, France). We measured the activity of nucleotide phosphodiesterase (NPP) 24 hours after transfection of HEK293 cells using pNP-TMP as substrate [7 (link),20 (link)]. Expression of human ENPP1 protein was detected by Western blot using a rabbit anti-human ENPP1 antibody, #5342, 1:1,000 (Cell Signaling, Boston, MA). An anti-human β-actin antibody, #4970, 1:1,000, was used to normalize protein loading (Cell Signaling). The cellular localization of the ENPP1 protein was analyzed in transfected COS7 cells using a monoclonal anti-human ENPP1 antibody, 1:100 (3E8, kind gift from Dr. Fabio Malavasi, Torino, Italy). Cells were stained with fluorescein isothiocyanate labeled phalloidin, #P5282, 1:40 (Sigma-Aldrich, Taufkirchen, Germany), to visualize plasma membrane localization. The PPi concentration in the medium of transfected HEK293 cells was measured 20 min after incubation with 20 μM GTP. PPi was quantified as previously described [10 (link),12 (link)].