Figure 2.

SAMHD1 nuclear import is mediated by KPNA2 and KPNB1. A) HeLa cells stably transduced with an MLV-vector encoding YFP-tagged wild type SAMHD1 were transiently transduced with Retro-SIREN-Q (Clontech) shRNA encoding vectors expressing two independent shRNAs for each karyopherin or shControl and subjected to autofluorescence microscopy 48 h after transduction. Nuclei were stained with Hoechst. All pictures were generated using the same microscope settings and analyzed using the same conditions. Scale bars, 50 μm. B) Cytoplasmic fluorescence was determined using software ImageJ for thirty cells for each slide shown in A) in an area of 535 pixels (total image size 1024x1024 pixels) of cytoplasm in proximity of the nucleus for each cell. Each dot represents measurement of cytoplasmic mean fluorescence intensities (MFIs) for one cell. Each cell was only counted once. C) GST-tagged wild type or SAMHD1_K11A encoding plasmids or the GST control were co-transfected with empty vector or HA-KPNA2 encoding vector in 293 T cells and GST-pull down (PD) was performed 48 h later. Tubulin served as the input loading control.