F1084

The expression of endonucleases in 293FT cells was measured using anti-FLAG antibodies, as all of the nucleases were tagged with the Flag epitope. Briefly, 2 × 10⁵ 293FT cells were seeded in 6-well plates and transfected with endonuclease vectors at a concentration of 500 ng/ml. Forty-eight hours after transfection, the cells were harvested, rinsed with phosphate-buffered saline and lysed in 150 μl of ice-cold RIPA buffer composed of 50 mM Tris, 150 mM NaCl, 0.5% Na deoxycholate, 1% Nonidet P-40 and 0.1% sodium dodecyl sulfate (SDS). Then the cell lysates were centrifuged at 4°C for 5 min at 8000 × g. The supernatant was subjected to electrophoresis using 8% SDS-polyacrylamide gel electrophoresis and then transferred onto the polyvinylidene difluoride (PVDF) membrane by performing electrobloting. The membrane was blocked in 5% non-fat milk in Tris-Buffered Saline with 0.1% Tween 20 (TBST) blocking solution at room temperature for 1 h and subsequently incubated with FLAG-specific monoclonal antibody diluted 1:1000 (F1084, Sigma) in TBST. The membrane was then subjected to a 1-h incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Zhongshan Jinqiao) at 1:1000 in TBST, followed by detection using the chemiluminescence labeling detection reagent ECL Plus (GE healthcare).

About 1×10⁷ HEK293T cells were transiently and separately transfected with 3×Flag-PHF14(1–914)-WT (wild type), 3×Flag-PHF14-ΔPZP (PZP domain was deleted), 3×Flag-PHF14-Δloop (insertion loop was replaced by ‘SS’ linker), and 3×Flag-PHF14-I386M (I386 was mutated to M) plasmids. At 72 h post-transfection, cells were washed with PBS and then lysed in 1.5 ml cold lysis buffer (Beyotime P0013 buffer with EDTA-free Selleck protease inhibitor). The cell lysates were then pre-cleared with pre-washed protein G agarose beads (Invitrogen,15920010) for 30 min. A total of 350 μl pre-clear lysate was incubated with 5 μl anti-H3 antibody (CST, 4620S) or anti-Flag antibody (Sigma, F1084) and 30 μl protein G agarose beads, or 5 μl corresponding IgG (CST, 3900S) and 30 μl protein G agarose beads overnight. Beads were then washed three times with lysis buffer, followed by boiling in sample buffer then loaded for gel electrophoresis and immunoblot. Anti-H3 and anti-Flag antibody used in immunoblot were obtained from ABclonal (A2348) and Sigma (F1804), respectively. All uncropped images can be found in Supplementary Figure S6D.