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Fig. 1 | Mobile DNA

Fig. 1

From: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation

Fig. 1

SAMHD1 T592A blocks LINE-1 but not HIV-1 replication in cycling cells. a 293T cells were transfected with a retrotransposition competent or a retrotransposition-defective (JM111) LINE-1 (L1)-GFP reporter plasmid and empty vector (pcDNA), SAMHD1 wt, non-phosphorylated SAMHD1 (T592A), phosphomimetic SAMHD1 (T592D), or an enzymatically inactive mutant (D207N). Five days posttransfection, GFP-positive cells were quantified by flow cytometry. The percentage of L1-GFP-positive cells is shown as average of triplicate transfections. Error bars represent the standard deviation. One out of three independent experiments is shown. Statistical analysis comparing control transfected cells with SAMHD1 expressing cells was done using one way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. b 293T cells were transfected with empty vector (pcDNA), SAMHD1 wt, non-phosphorylated SAMHD1 T592A, or the enzymatically inactive mutant (D207N). Two days posttransfection, cells were infected with VSV-G-pseudotyped HIV-GFP reporter virus at the indicated MOIs. Three days later, GFP-positive cells were analyzed by flow cytometry. The percentage of HIV-GFP-positive cells is depicted as average of triplicate infections with error bars indicating the standard deviation. One out of three independent experiments is shown. c 293T cells were transfected with the indicated SAMHD1-myc expressing vectors. Lysates were analyzed 2 days posttransfection by immunoblot. Membranes were probed with phosphoT592-specific, myc-specific, and HRP-containing corresponding secondary antibodies. One out of three independent experiments is shown. d 293T shC or shSAMHD1 cells were transfected with L1-GFP reporter plasmid. GFP-positive cells were quantified 5 days posttransfection by flow cytometry. The percentage of L1-GFP-positive cells is shown as average of triplicate transfections. Error bars represent the standard deviation. Statistical analysis was done using an unpaired, two-tailed student’s t test. ns, not significant. The shRNA-mediated knockdown and the phosphorylation status of SAMHD1 were analyzed by immunoblot with a SAMHD1-specific and a phosphoT592-specific antibody. One out of three independent experiments is shown

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