Bioruptor power up

Endogenous co-IPs were performed on nuclear extracts of WT-1 cells. Briefly, nuclei were obtained upon cell lysis in cytosolic lysis buffer [10 mM tris (pH 7.9), 10 mM NaCl, 0.1 mM EDTA (pH 8), and 0.1 mM EGTA] and extracted in nuclei lysis buffer [20 mM tris (pH 7.9), 400 mM NaCl, 1 mM EDTA (pH 8), and 1 mM EGTA]. Nuclear extracts were then sonicated on a Bioruptor Power-Up (Diagenode), centrifuged at 14,000g for 20 min at 4°C, and precleared with Protein A/G magnetic beads (Thermo Fisher Scientific) for 2 hours at 4°C. Immunoprecipitation of 2 mg (for Prdm16 IP) and 1 mg (for LaminB IP) of the nuclear extract was then carried out overnight at 4°C with the specified antibodies [15 μg of anti-Prdm16 (Abcam, ab106410), 5 μg of anti-LaminB (Abcam, ab16048), and corresponding equal amounts of normal rabbit IgG (Santa Cruz Biotechnology) were used as negative IP controls]. Beads were then added for 2 hours to recover immunocomplexes and then washed six times with IP buffer [50 mM tris (pH 7.9), 150 mM NaCl, 1 mM EDTA (pH 8), and 1 mM EGTA]. Immunocomplexes were eluted with 25 μl of Laemmli sample buffer (4×) and heated at 95°C for 10 min before SDS–polyacrylamide gel electrophoresis (PAGE).