Ab191387

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.

Cells were collected by detachment with trypsin and then lysed in radio immunoprecipitation assay lysis buffer (Boster, Hubei, China), followed by estimation of protein concentration using a bicinchoninic acid quantification kit (Boster). Subsequent to separation using freshly-prepared 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the protein was electro-transferred onto polyvinylidene fluoride membranes. The membrane underwent 5% bovine serum albumin (BSA) blocking and probing with primary antibodies (Abcam, Cambridge, UK) to KDM2A (ab191387, 1:1000), JAG1 (ab7771, 1:500) and GAPDH (ab181602, 1:500). Following incubation with secondary antibody goat anti-rabbit immunoglobulin G (IgG) (ab205719, 1:500, Abcam), immunoblots were visualized with enhanced chemiluminescence detection reagents (Millipore Corp., Bedford, MA, USA) and then captured under the Bio-Rad image system (Bio-Rad, Hercules, CA, USA). Gray values of target protein bands were quantified using Image J software, with GAPDH as a normalizer.