Fig. 6

SAMHD1 T592A does not affect LINE-1 expression. a 293T cells were transfected with a L1 promoter construct driving the luciferase reporter gene expression (L1 promoter-Luc) together with empty vector (pcDNA), SAMHD1 wt protein, or the indicated mutants. Two days post transfection, cells were lysed and luciferase activity (relative light units, RLU) was determined in quadruplicates. The average RLU/s of three independent transfections is shown. Error bars represent the standard deviation of the mean. b 293T cells were transfected with the L1 reporter plasmid pAD2TE1 together with control plasmid or expression plasmids for SAMHD1 T592A, D207N, or ZAP. Total mRNA was isolated at 12, 24, 36, and 48 h posttransfection and analyzed by qRT-PCR with oligonucleotides specific for the T7 tag sequence fused to ORF1 within the transcripts of transfected L1. The average number of L1 copies per 100 ng RNA input from three independent experiments is plotted. For better comparison, all values were normalized on L1 transcripts present in T592A expressing cells 36 h posttransfection. Error bars indicate the standard deviation. Statistical analysis was done using one way ANOVA followed by Tukey’s multiple comparison test. ns, not significant. Two days posttransfection, protein expression was analyzed by immunoblot using T7- (ORF1p), myc- (SAMHD1), and HA-specific antibodies. c and d 293T cells were transfected with the L1 reporter plasmid pAD2TE1 encoding T7-tagged ORF1p or an ORF2p-3xFLAG expression plasmid together with plasmids encoding GFP, or the human SAMHD1 proteins wt SAMHD1, T592A, T592D, or D207N. Two days posttransfection, immunoblot analysis was performed to analyze SAMHD1-myc, ORF1p-T7, and ORF2p-FLAG expression levels. e and f 293T cells were transfected with the L1 reporter plasmid pAD2TE1 encoding ORF1p-T7 or ORF2-3xFLAG expression plasmid and increasing amounts of SAMHD1 T592A or D207N. To ensure a constant amount of transfected DNA, GFP expression plasmid was added to the transfection reactions. Immunoblot analysis was performed 2 days posttransfection to determine SAMHD1-myc, ORF1p-T7, or ORF2p-FLAG expression levels. One of at least three independent experiments is shown