Fig 3. Association of LINE-1 RNP with stress granule marker G3BP1.

(A) Schematic illustration of the immunoprecipitation/RT-PCR protocol. LINE-1 RNA that was co-immunoprecipitated (IP) was measured by nest RT-PCR. The PCR products were analyzed by gel electrophoresis followed by extraction from the gel and sequencing. (B) Depiction of the full-length LINE-1 RNA (FL1) and the spliced ORF2 transcript (SpORF2). Arrows indicate the position of the primers used in RT-PCR. This primer pair amplifies a 304 bp sequence of the SpORF2 RNA. (C) HeLa cells were transfected with Flag-ORF1p and CMV-L1-neo^RT. Immunoprecipitation was performed with anti-Flag M2 antibodies to pull down Flag-ORF1p as well as ORF1p expressed from CMV-L1-neo^RT. The immunoprecipitated materials were treated with or without RNase A during IP as indicated. Presence in the precipitated materials of ORF1p and G3BP1 was determined by western blotting. RT-PCR was performed to measure the levels of LINE-1 RNA in the precipitated samples. The PCR products were analyzed by gel electrophoresis. (D) HeLa cells were co-transfected with Myc-G3BP1 and CMV-L1-neo^RT. G3BP1 was precipitated with anti-Myc antibody. The immunoprecipitated materials were treated with or without RNase A during IP as indicated. Co-immunoprecipitation of ORF1p and LINE-1 RNA was determined by western blotting and RT-PCR, respectively. The PCR products were analyzed by gel electrophoresis. The PCR products were also extracted from the gel and sequenced to confirm their being LINE-1 sequence.