Figure 5.

Analysis of dNTPase activity in SAMHD1 splice variants. (A) HeLa cells were transfected with pcDNA-HA-SAMHD1 WT, Δ14, Δ8-9 or a catalytically inactive SAMHD1 mutant, H206A/D207A (AA). Amounts of transfected DNA were adjusted to achieve comparable protein levels. Cell extracts were prepared 24 h later and used for immunoprecipitation with HA-beads as described in Methods. 15% of the total immunoprecipitated SAMHD1 proteins were separated on SDS-PAGE and detected by immunoblot using SAMHD1 specific antiserum. (B) The remaining immunoprecipitated protein was used in a dTTPase assay directly on the beads using 0.5 μCi α-³²P] dTTP and conditions reported by Lahouassa et al.[21] and described in Methods. Where indicated, 200 μM dGTP was added to the reaction buffer. Released triphosphate (PPP) was separated by polyethyleneimine (PEI)-cellulose thin layer chromatography using 0.8 M LiCl as the mobile phase.