Ne per nuclear cytoplasmic extraction kit

Cytoplasm and nuclear fractionation were performed according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). In brief, cells were harvested and washed once with cold phosphate-buffered saline (PBS) (pH 7.4) containing 19 g of NaCl, 0.3 g of Na₂HPO₄·2H₂O, and 6 g of Na₂HPO₄·12H₂O for a total of 1 L. The cells were then lysed with the help of NE-PER Nuclear & Cytoplasmic extraction kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The cytoplasmic and nuclear fractionation samples were stored at −80°C in preparation for Western blotting detection.

Ex vivo RT activity in nuclear and cytoplasmic protein fractions was assessed using an Alu RNA–templated reaction. Nuclear and cytoplasmic fractionation was prepared using an NE-PER nuclear cytoplasmic extraction kit (Thermo Fisher Scientific), as per the manufacturer’s instructions. Briefly, in this assay, exogenous Alu RNA and Alu-R primer (5′- ACCTCCCGGGTTCACGCCATT-3′) were incubated with nuclear or cytoplasmic protein lysate containing endogenous L1 ORF2p, which interacts with Alu RNA in trans giving rise to an Alu cDNA. The RT reaction was carried out in a 20-μl reaction mix containing Alu RNA template (10 ng), Alu primer (10 pmol), dNTPs mix, cytoplasmic or nuclear protein, and Quantiscript RT Buffer (Qiagen). The reaction mixture was incubated at 42°C for 30 min. The resulting cDNA was quantified by qPCR using Alu RNA template-specific primers. Heat denaturation of nuclear or cytoplasmic fraction was performed by heat inactivation at 95°C for 10 min.