Fig. 4

PP2A-B55α holoenzymes interact with SAMHD1 in cycling cells. a SAMHD1 interacts with PP2A-B55α holoenzymes. HEK293T cells were co-transfected with constructs expressing N-terminally FLAG-tagged SAMHD1 (3.5 µg) and different N-terminally GST-tagged PP2A B-type subunits (7.5 µg). 48 h post-transfection, cells were harvested, lysed and CoIPs performed using anti-GST-coated magnetic beads. Proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. Data shown are representative of three independent experiments. b Reciprocal CoIP experiment. HEK293T cells were co-transfected with constructs expressing N-terminally FLAG-tagged SAMHD1 (5.5 µg) and GFP-tagged PP2A B55α subunit (5.5 µg). Sole transfection of the expression plasmid encoding GFP-tagged PP2A B55α subunit was used as a negative control. 48 h post-transfection, cells were harvested, lysed and CoIPs performed using anti-FLAG-coated agarose beads. Proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. Data shown are representative of three independent experiments. c SAMHD1 interacts with endogenous PP2A B55α subunit in HEK293T cells. Cells were transfected with constructs expressing N-terminally FLAG-tagged SAMHD1 or empty vector as a negative control. 48 h post-transfection, cells were harvested, lysed and CoIPs performed using anti-FLAG-coated agarose beads. Proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. Data shown are representative of two independent experiments. d Endogenously expressed SAMHD1 interacts with endogenous PP2A B55α subunit in monocytic THP-1 cells. Cycling THP-1 cells were harvested (2 × 10⁷ cells/sample), lysed and CoIPs performed using anti-IgG1/SAMHD1-coated sepharose beads. Proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. Data shown are representative of two independent experiments. e Comparison of basic amino acids in the C-terminal region of human (Hs) and murine (isoform 1; Mm) SAMHD1. Alignment of SAMHD1 protein sequences was generated with ClustalW. Basic residues are highlighted in black, while non-basic residues at the corresponding positions are highlighted in gray. Introduction of non-basic residues into human SAMHD1 (Hs∙Mm) and, vice versa, basic residues into murine SAMHD1 (Mm∙Hs) are marked in red. f SAMHD1 interacts with PP2A-B55α holoenzymes through basic amino acids. HEK293T cells were transfected with constructs expressing N-terminally FLAG-tagged human/murine (isoform 1) SAMHD1 or mutants with substituted/introduced basic residues. Empty vector was transfected as a negative control. 48 h post-transfection, cells were harvested, lysed and CoIPs performed using anti-FLAG-coated agarose beads. Proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. Data shown are representative of three independent experiments