Fig. 3

Murine SAMHD1 is phosphorylated at threonine 603 in cycling cells. a Schematic overview of the different SAMHD1 constructs used in the study. Human SAMHD1 and both murine isoforms contain the SAM domain and the enzymatic active HD Domain. Phosphorylation of threonine 592 in human SAMHD1 regulates its antiviral activity. The corresponding threonine is at position 603 in murine isoform 1 (iso1), but is absent in the isoform 2 (iso2) sequence due to differential splicing. b 293T cells were co-transfected with expression plasmids for 3′myc-tagged human SAMHD1, human SAMHD1-mutant T592A (T592A), mouse iso1, iso1-mutant T603A (T603A), or murine iso2. After 2 days, cell lysates were prepared and analyzed on an immunoblot probed with anti-myc MAb, or anti-pSAMHD1-T592 antibody. c THP-1 huSAMHD1 KO cells stably expressing 3′myc-tagged murine SAMHD1 isoform 1 (iso1), iso1-T603A, or iso1-T603D were incubated with 50 nM PMA (+PMA) or without PMA (−PMA) for 16 h. Cell lysates were separated by regular SDS-PAGE (-Phos-tag) or on a 8 % polyacrylamide gel containing 25 mM Phos-tag reagent (+Phos-tag). Upon transfer, membranes were probed with an anti-myc MAb. Asterisk marks the slower migrating bands in the Phos-tag gel. d Cycling THP-1 cells expressing 3′myc-tagged iso1 were incubated for 14 h with DMSO or one of the CDK inhibitors roscovitine (Rosco), olomoucine II (Olo II), or CDK2-specific inhibitor (CDK2-I) at 0.3 µM or 3.0 µM. After 14 h, cell lysates were separated by SDS-PAGE and analyzed by immunoblot using anti-myc MAb, anti-pSAMHD1-T592 antibody, or anti-HSP90 antibody