SAMHD1 increases LINE-1 RNP sequestration in stress granules.

<p>(A) HeLa cells were co-transfected with CMV-L1-neo^RT DNA and wild type Myc-SAMHD1 or SAMHD1-H233A mutant DNA. Endogenous G3BP1 was precipitated with anti-G3BP1 antibody. The anti-rabbit IgG was utilized as a control for non-specific binding. Co-immunoprecipitation of ORF1 and LINE-1 RNA was determined by western blotting and nest RT-PCR, respectively. Intensities of ORF1 bands (western blotting) and LINE-1 RNA bands (nest RT-PCR) were quantified using the ImageJ automated digitizing program (NIH). The results from three independent experiments are summarized in the bar graph. Levels of ORF1 and LINE-1 RNA in the control (SAMHD1 (-), G3BP1 antibody (+)) are arbitrarily set as 1. (B) Co-immunoprecipitation of LINE-1 RNA was determined by real-time quantitative PCR as described in Materials and Methods. The results from three independent experiments are summarized in the bar graph. Levels of ORF1 and LINE-1 RNA in the control (SAMHD1 (-), G3BP1 antibody (+)) are arbitrarily set as 1. (C) HeLa cells were co-transfected with Myc-KPNA2, Flag-ORF1 with or without HA-SAMDH1. Immunoprecipitation was performed with anti-Flag M2 gel. Presence of Myc-KPNA2 in the precipitated materials was determined by western blotting. Intensities of the KPNA2 bands were quantified using the ImageJ automated digitizing program (NIH), the results from three independent experiments are summarized in the bar graph. Level of KPNA2 in the control cells is arbitrarily set as 1. (D) SAMHD1 is not associated with ORF1. HeLa cells were co-transfected with Myc-SAMHD1 DNA and ORF1-Flag DNA. Myc-SAMHD1 was immunoprecipitated with anti-Myc antibody. The presence of ORF1 in the precipitated materials was examined by western blotting. * indicates p<0.05, ns denotes “not significant”.