Ezh2 antibody

Cells were collected at a concentration of 30 μg/sample for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) using a pre-chilled radioimmunoprecipitation assay buffer cocktail with a phosphatase inhibitor. The polyvinylidene fluoride membrane (0.45 μm) was transferred to 1 × Tris-buffered saline solution with Tween-20 detergent (TBST) to block 2% bovine serum albumin. Then, the primary EZH2 antibody (#4905; Cell Signaling Technology) was cultured at 4°C (1:1,000) for 12 h, and the secondary antibody at 37°C for 1 h. After four rounds of TBST washing, the membrane was colored by chemiluminescence using the ECL Western Blotting Substrate Kit (ab65623; Abcam, Cambridge, MA, United States).

HUVEC were grown on gelatin (#TC041, HiMedia)-coated coverslips up to 70% confluency. After completion of treatment, they were washed with PBS and subjected to fixation with 4% ice-cold paraformaldehyde. Cells were incubated with 0.1% Triton X for permeabilization. Cells were then incubated with BSA (1%) for 1 h, followed by overnight incubation with EZH2 antibody (1:1000, #5246, Cell Signaling Technology). Cells were washed with PBS to remove unbound primary antibody, and were subsequently incubated with Alexa fluor 555 secondary antibody (1:4000; #A32732, Thermo Fisher Scientific) for 2 h. To stain F-actin, cells were incubated with rhodamine-tagged phalloidin (1:5000; #R415, Thermo Fisher Scientific) for 30 min, followed by incubation with DAPI (#D9542, Sigma-Aldrich) for nuclear staining. Fluorescence images were captured using a Zeiss ApoTome.2 microscope (Carl Zeiss, Jena, Germany), and intensities were measured using ImageJ software.

Cells were collected on ice and lysed in 0.2% NP‐40 co‐IP buffer (10 × 10^‐3m Tris‐Cl, pH 7.4, 150 × 10^‐3m NaCl, 0.2% NP‐40, and 10% glycerol) supplemented with protease inhibitors and protein phosphatase inhibitor cocktail, incubated on ice for 15 min, and cleared by centrifugation at 13 200 rpm at 4 °C for 15 min. Total protein lysate (2–5 mg) was precleared with normal rabbit IgG for 2 h at 4 °C, and 1 mL precleared lysate was subjected to immunoprecipitation with EZH2 antibody (Cell Signaling Technology#5246) or MYPT1 antibody (Cell Signaling Technology #8574) as well as protein A/G agarose beads overnight at 4 °C. The immunoprecipitates were washed six times with co‐IP buffer, and then the washing buffer was completely removed from the beads. The beads were boiled in SDS sample buffer, and then the proteins were separated by SDS‐PAGE and transferred to nitrocellulose membranes. The blots were developed with anti‐MYPT1 (1:1000, Santa Cruz, sc‐514261) or with anti‐EZH2 (Cell Signaling Technology #5246 & #3147) antibodies.