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. Author manuscript; available in PMC: 2013 Nov 15.
Published in final edited form as: Nat Med. 2012 Nov;18(11):10.1038/nm.2964. doi: 10.1038/nm.2964

Figure 2.

Figure 2

SAMHD1 is expressed in resting CD4+ T cells and depleted by Vpx. (a) RT-PCR–based quantification of SAMHD1 mRNA levels in CD4+ T cells. RNaseP-normalized mRNA levels in resting and PHA- and IL-2-activated CD4+ T cells are presented relative to those from THP-1 cells that were arbitrarily set to 100%. Means + s.e.m. of four donors. (b) Immunoblotting for endogenous expression of SAMHD1 in CD4+ T cells (two donors). GAPDH: loading control. (c) In situ expression of SAMHD1 in explants of human tonsil. Left and center, immunohistochemical detection of SAMHD1. Nuclei were counterstained with hematoxylin. GC, germinal center; LW, lymphocyte wall; PR, perifollicular region. Right, immunofluorescence analysis of SAMHD1 expression (green) relative to the proliferation marker Ki67 (red). (d) SAMHD1 immunoblot analysis of a nuclear-cytoplasmic fractionation of resting CD4+ T cells. WCL, whole cell lysate. GAPDH and B23 served as cytoplasmic and nuclear markers, respectively. (e) Three-dimensional reconstructions of deconvolution confocal microscopy images of a resting CD4+ T cell co-stained for SAMHD1 (green) and the nuclear envelope protein lamin A (red). At right is an optical section across the cell to show nuclear and cytoplasmic SAMHD1. (f) Immunoblots of endogenous SAMHD1 in nucleofected resting CD4+ T cells expressing WT Vpx, Vpx Q76A or an empty vector control. Co-expression of surface-exposed Display-GFP was used to purify a homogenous population of nucleofected cells. Similar results were obtained for two other donors. (g) Quantification of intracellular SAMHD1 amounts in nucleofected resting CD4+ T cells. Cells were nucleofected to co-express GFP together with WT Vpx, Vpx Q76A or an empty control and 16 h later assessed for SAMHD1 expression in relation to GFP by flow cytometry.