Fig. 3

PP2A, not PP1, is able to dephosphorylate SAMHD1 pT592 in vitro. a Targeted nano-LC-MS/MS analysis reveals PP2A as responsible phosphatase acting on SAMHD1 pT592. 1 µg of recombinant SAMHD1 was incubated with equilibrated units of PP2A_D and PP1, or buffer alone (control) for 1 h at 30 °C. After stopping the reaction by addition of 5 × SDS-PAGE sample buffer, the 1D-gel piece containing SAMHD1 was subjected to trypsin digestion followed by targeted nano-LC-MS/MS of the resulting peptides. Relative quantification of phosphorylated and non-phosphorylated peptides encompassing T592 in different conditions (treatment with PP2A_D or PP1 compared to buffer only (control)) is displayed by means of their SRM intensity ratios. b Validation of MS data by immunoblot analysis with anti-SAMHD1 pT592 antibodies, executed on the same samples as described in Fig. 3a. The data are representative of three independent experiments. c Time- and concentration-dependency of SAMHD1 pT592 dephosphorylation by PP2A_D. In vitro-dephosphorylation of recombinant SAMHD1 was executed with the indicated units of PP1 or PP2A_D for the indicated time points. When appropriate, 50 nM OA was added to the phosphatases in a 10 min-pre-incubation step, before addition of the substrate, and dephosphorylation for the indicated times. SAMHD1 (de)phosphorylation was monitored by immunoblotting of samples with SAMHD1 pT592-specific antibodies