Esl103

For co-immunoprecipitation reactions, we used Nuclear Complex Co-IP Kit (Active Motif54001) according to the manufacturer’s recommendations. Briefly, 200 µg of nuclear extract was used per IP reaction and incubated with 3 µg of the corresponding antibody. Some of the nuclear extracts were treated with 125 U/mL of benzonase (Millipore-Sigma, 71205-M) on ice for 90 min. The antibody/extract mixture was incubated overnight at 4°C on a rotator. Protein G beads were added to each IP reaction. We used IP buffers from the Kit, containing low salt and detergent. Following the IP, 2X sample buffer was added to each IP reaction; samples were boiled and run on an SDS-PAGE gel. The antibodies used for Co-IP and detection: custom-designed mouse monoclonal antibody against BORIS, mouse monoclonal antibody against CTCF (Santa Cruz, # sc-271514), rabbit polyclonal antibody against SRCAP (Kerafast, ESL103), Rabbit IgG (Abcam, ab37415), and p400 (Novus, NB200-210). Immunoprecipitated samples were resolved by SDS-PAGE, transferred to a PVDF membrane, and incubated with the indicated antibodies. Detections were performed using ECL reagents.

Protein extracts were prepared with RIPA Lysis buffer (Millipore) containing 50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 500 mM NaCl, 1 mM EDTA, and 1 × protease inhibitor cocktail (Roche Applied Science). To detect LGALS3BP secreted protein, cell supernatant with trichloroacetic acid precipitation was prepared according to protocol [113 (link)]. The protein extracts were resolved by SDS-PAGE, transferred to a PVDF membrane, and incubated with the indicated antibodies. Detections were performed using ECL reagents. The antibodies used are as follows: custom-designed mouse monoclonal antibody against BORIS, mouse monoclonal antibody against CTCF (Santa Cruz, sc-271514), mouse monoclonal antibody against Tubulin (Abcam, ab7291), rabbit polyclonal antibody against SRCAP (Kerafast, ESL103), Rabbit IgG (Abcam, ab37415), rabbit monoclonal against histone H3 (Abcam, ab201456), rabbit monoclonal against LINE-1 ORF1p (Abcam, ab216324), rabbit monoclonal against IRF7 (Thermo Fisher Scientific, SC0617), rabbit polyclonal against LGALS3BP (Thermo Fisher Scientific, PA5-79597), rabbit polyclonal against IFIT1 (Abcam, ab236256), rabbit polyclonal against IFI44 (Thermo Fisher Scientific, PA5-96967), rabbit polyclonal against Aim2 (Cell Signaling, #63660), rabbit polyclonal against OAS1 (MyBioSource, MBS129033).