Fig. 5
PP2A-B55α holoenzymes dephosphorylate SAMHD1 at T592 in vitro and in cells. a–c PP2A-B55α holoenzymes dephosphorylate SAMHD1 at pT592 in vitro. GFP-trapped active PP2A trimer complexes or GFP only were retrieved from HEK293T cells. After incubation with recombinant SAMHD1 for the indicated time points, samples were assessed by immunoblotting using antibodies specific to the indicated proteins. a Before incubation with recombinant SAMHD1, undiluted (1/1) and 1/3 diluted beads were either pre-incubated with buffer or 50 nM okadaic acid (OA) for 10 min at 30 °C. Data are representative of two independent experiments. b Purified PP2AD was added as an additional condition. Anti-PP2A C immunoblots allow to directly compare the amount of C subunit in both PP2A complexes (PP2A-B55α trimer versus PP2AD). The lanes shown are derived from the same blot, but were not adjacently loaded. Data are representative of two independent experiments. c Anti-C immunoblotting indicates the amount of retrieved PP2A C subunit in these complexes. Data are representative of two independent experiments. d, e Inhibition of PP2A by OA (d) or siRNA-mediated silencing (e) increases SAMHD1 phosphorylation at T592 in cycling HeLa cells. d HeLa cells were treated with the phosphatase inhibitor OA (2 nM), harvested at different time points and analyzed by immunoblotting. e HeLa cells were transfected with control siRNA (lane 1), simultaneously with three different siRNAs targeting all subunits of the PP2A-B55α trimer (lane 3) or with single siRNAs targeting individual PP2A subunits (lane 4–6). 52 h post-transfection, cells were harvested and analyzed by immunoblotting. Data shown are representative of three (d) or two (e) independent experiments, respectively. f, g Inhibition of PP2A by OA (f) or by siRNA-mediated silencing of PP2A B55α subunit (g) increases SAMHD1 phosphorylation at T592 in MDMs. f MDMs were treated with the phosphatase inhibitor OA (2 nM), harvested at different time points and analyzed by immunoblotting. Data shown represent three donors analyzed. g MDMs were transfected with control siRNA or siRNA targeting B55α. 48 h post-transfection, cells were harvested and whole-cell lysates were analyzed by immunoblotting (g, left panel). For quantification, the signal of PP2A B55α subunit was normalized to GAPDH and compared to the control (g, left panel). In parallel, RNA samples were collected and PPP2R2A (B55α) mRNA levels determined by RT-qPCR (g, right panel). Data were normalized to the reference gene RPL13A. Fold change of PPP2R2A (B55α) mRNA level to siCtr was calculated based on three technical replicates; the graph shows the mean ± SD of the technical replicates (right panel). Data shown represent one of three donors analyzed
