Fig. 2

SAMHD1 T592A inhibits LINE-1, IAP, MusD, and AluY reporter elements in HeLa cells. a and b HeLa cells were transfected with a L1-neomycin reporter plasmid and either empty vector (pcDNA), SAMHD1 wt, or the indicated SAMHD1 mutants using Lipofectamine. Cells were incubated with G418-containing medium for 10 days to select for successful retrotransposition events and probed with crystal violet. b Crystal violet stained G418-resistant foci of L1-transfected HeLa cells. c HeLa cells were transfected with IAP-neomycin reporter plasmid and either empty vector (pcDNA) or the indicated SAMHD1 mutants. d HeLa cells were transfected with MusD-neomycin reporter plasmid and either empty vector (pcDNA) or the indicated SAMHD1 mutants. e HeLa HA cells were transfected with an AluY-neomycin reporter plasmid, an L1 ORF2p expression construct, and either empty vector (pcDNA), SAMHD1 wt, SAMHD1 T592A, or SAMHD1 D207N. In general, G418-resistant foci were analyzed by crystal violet staining 10 days post selection using ImageJ software. The number of resistant foci is shown as average of triplicate transfections with error bars indicating the standard deviation. Statistical analysis comparing control transfected cells and SAMHD1 expressing cells was done using one way ANOVA followed by Tukey’s multiple comparison test. *** p < 0.001; ns, not significant. f HeLa cells were transfected with the indicated SAMHD1-myc expression plasmids. Lysates were analyzed 2 days posttransfection by immunoblot. Membranes were probed with phosphoT592-specific and myc-specific primary antibodies followed by HRP-containing secondary antibodies. One of three independent experiments is shown