Figure 2From: Contribution of oligomerization to the anti-HIV-1 properties of SAMHD1Oligomerization, RNA binding and intracellular distribution of SAMHD1 variants. (A) Oligomerization of SAMHD1 variants was tested as previously described [31]. Briefly, human 293Â T cells were co-transfected with a plasmid expressing wild type SAMHD1-HA and a plasmid either expressing wild type or mutant SAMHD1-FLAG proteins. Cells were lysed 24Â hours after transfection and analyzed by Western blotting using anti-HA and anti-FLAG antibodies (Input). Subsequently, lysates were immunoprecipitated by using anti-FLAG agarose beads. Anti-FLAG agarose beads were eluted using FLAG peptide, and elutions were analyzed by Western blotting using anti-HA and anti-FLAG antibodies (Immunoprecipitation). Similar results were obtained in two independent experiments and representative data is shown. WB, Western blot; IP, Immunoprecipitation; WT, wild type. (B) Similar immunoprecipitations were performed by pulling down an HA-tagged variant with its corresponding FLAG-tagged variant. (C) The ability of SAMHD1 variants to bind nucleic acids was tested as previously described [31]. Human 293Â T cells were transfected with plasmids expressing the SAMHD1 variants were lysed (Input) and incubated with the RNA analog ISD-PS immobilized to Strep Tactin Superflow affinity resin. Eluted proteins from the resin were visualized by Western blotting using anti-FLAG antibodies (Bound). Similar results were obtained in three independent experiments and a representative experiment is shown. ISD-PS, interferon-stimulatory DNA sequence containing a phosphorothioate backbone. (D) Intracellular distribution of SAMHD1 variants in HeLa cells. HeLa cells expressing the indicated SAMHD1-FLAG variants were fixed and immunostained using antibodies against FLAG (red) as previously described [31, 44]. Cellular nuclei were stained by using DAPI (blue). Image quantification for three independent experiments is shown in Additional file 1.Back to article page