DsDNA Binding Assay and Co-immunoprecipitation

The 5′-biotin-labeled 45-bp dsDNA was also synthesized and obtained from GENEWIZ (Shouzhou, China). Streptavidin Agarose (Cat No: S951, ThermoFisher Scientific) was washed three to five times with PBS by centrifugation at 10,000 g and suspended in PBS. Each milliliter of streptavidin agarose was mixed with 20 nmol 5′-biotin-labeled 45-bp dsDNA, incubated at RT for 30 min, and washed three to five times with PBS, and the resultant 45-bp dsDNA-agarose was stored at 4°C for protein pull-down assay. For protein pull-down assay, cells in a six-well plate (8 × 10⁵ cells/well) were transfected for 48 h, harvested, and lysed in 500μl of RIPA buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 1% sodium deoxycholate, and 1% Triton X-100) containing protease inhibitors on ice for 30 min. The 50 μl of cleared lysate was used as input control, and the remainder was incubated with 20 μl of dsDNA-agarose at 4°C overnight with shaking. Next day, the dsDNA agarose was washed three times by centrifugation, and bound proteins were eluted with 20 μl of 2 × SDS sample buffer by heating at 100°C for 10 min. The elution supernatants from centrifugation together with input controls were subjected to Western blot analysis. For co-immunoprecipitation, the cleared cell lysate from transfected cells was incubated with 1 μg of specific antibody at 4°C overnight with shaking and further incubated with Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology) for 2–3 h. The agarose was similarly washed and eluted with 20 μl of 2 × SDS sample buffer. The elution and input were both subjected to Western blot analysis.