Ab176920

Whole cells or human endometrial tissues were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) including a protease inhibitor cocktail (ThermoFisher Scientific). About 20 μg protein extract were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight. Next day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The membranes were visualized using Chemiluminescent HRP Substrate (Millipore). The immunoreactive protein band density was quantified using Image J software (NIH). Raw data of all western blots are provided in Supplementary Fig. 4.

Oocytes were fixed for 1 h in 4% formaldehyde in a phosphate buffered saline (PBS, pH 7.4) at room temperature (RT). After permeabilization with 0.2% Triton X-100 for 20 min, oocytes were incubated with primary antibodies diluted in blocking solution overnight at 4 °C, including Kdm2a (Abcam, ab191387, 1:500), H3K36me1 (Abcam, ab176920, 1:1000), H3K36me2 (Abcam, ab176921, 1:1000), and H3K36me3 (Abcam, ab9050, 1:1000). After three washes with PBS, oocytes were incubated with corresponding secondary antibody at RT for 30 min, and stained with propidium iodide (PI, 1 μg/mL in PBS) for 5 min. Labeled oocytes were mounted on slides and obtained images using a Zeiss LSM800 confocal microscope (Carl Zeiss, Germany) with the same parameters. The ZEN imaging and ImageJ software were used to analyze the images.