Ultranuclease

DNA sequences of HelB–EGFP, EGFP–BLM, EGFP–RMI1, EGFP–WRN, EGFP–ATRIP, EGFP–ETAA1, RAD9–EGFP, or EGFP–MRE11 were cloned into the plasmid pcDNA3.1(+). 48 h after transfection, HEK 293T cells were washed with phosphate-buffered saline (PBS) and lysed in IP buffer comprising 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 5% w/v glycerol, ultraNuclease (Yeasen), and 1×PMSF and protease inhibitor cocktail (Topscience). For IP reactions, cleared cell lysates were incubated with anti-GFP magnetic beads (Beyotime) for 4 h at 4 °C with rotation. The beads were washed three times with tris buffered saline (TBS) using a magnetic separator. The bound proteins were eluted with 50 μl 5×SDS-loading buffer. Samples were boiled at 95 °C for 5 min and separated with 10% SDS-PAGE gels for immunoblot analysis.

Flag-tagged PTPN21 (1 μg/μl; WT or mutant) plasmids alone or in the presence of HA-tagged 18E7 plasmids (0.25 μg/μl) were tranfected into HEK293T cells. Forty-eight hours after transfection, cells were washed twice with ice-cold PBS and lysed in 250 μl of ice-cold lysis buffer [20 mM tris-HCl (pH 8.0), 0.15 M NaCl, 10% glycerol, and 0.5% NP-40], supplemented with EDTA-free protease inhibitors (Topscience, catalog no. C0001), phosphatase inhibitors (Topscience, catalog no. C0004), and UltraNuclease (Yeasen Biotechnology, catalog no. 20156ES). Cell lysates were centrifuged at 700g for 5 min, and the supernatants were incubated with Flag beads at 4°C for 4 hours. The beads were washed three times with lysis buffer, mixed with 1× sample loading buffer, and boiled at 95°C for 5 min for Western blotting.