Fig. 3

Human SAMHD1 mutants differentially inhibit LINE-1 retrotransposition. a 293T cells were transfected with L1-GFP reporter plasmid or the control vector JM111, together with empty vector (pcDNA), SAMHD1 wt, non-phosphorylated SAMHD1 T592A, the phosphomimetic variant SAMHD1 T592D, the HD domain mutants SAMHD1 D207N and D311N, the RNase-defective mutant Q548A, the allosteric site mutant D137A, or the oligomerization-defective mutant L428S/Y432S. The mean percentage of L1-GFP-positive cells of three independent experiments normalized on pcDNA transfected control cells is shown. Error bars represent the standard deviation of the mean. b 293T cells were transfected with L1-GFP reporter plasmid or control vector (JM111) and either empty vector (pcDNA), SAMHD1 wt, or the different SAMHD1 mutants shown in (a) but in a SAMHD1 T592A background. Five days posttransfection GFP-positive cells were quantified by flow cytometry. The percentage of L1-GFP positive cells is plotted as average of triplicate transfections. Error bars indicate the standard deviation. For western blot analysis, SAMHD1 protein expression was analyzed by immunoblot using phosphoT592-specific or myc-specific antibodies 2 days posttransfection. One out of three independent experiments is shown. For statistical analysis, one way ANOVA followed by Tukey’s multiple comparison test was used. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant