H3k27me1

For the myenteric plexus, fresh gastric tissues were placed in pre-cooled phosphate-buffered saline (PBS) solution. Thereafter, the mucosa and muscularis tissues were separated and fixed in 4% paraformaldehyde for 10 min. The paraffin-embedded gastric tissue sections were dewaxed and hydrated and subjected to antigen retrieval. The tissues were incubated with donkey serum containing 0.3% Triton X‐100 at 4 °C overnight for blocking of nonspecific binding. Subsequently, the tissue sections were incubated overnight at 4 °C with the specific primary antibodies: GFAP (ABclonal, Wuhan, China), HuC/D (Abcam, Cambridge, UK), S100B (Abcam, Cambridge, UK), β-Tubulin (ABclonal, Wuhan, China) H3K9me1 (A2358, ABclonal), H3K9me3 (A2360, ABclonal), H3K27me1 (A2361, ABclonal), and H3K27me3 (A2363, ABclonal). After washing three times with PBS, the preparations were then stained with the secondary antibody and incubated for 2 h at room temperature. The cell nuclei were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) for 20 min. The specimens were observed using a confocal laser scanning microscope (Olympus, Tokyo, Japan).

The total proteins and nuclear proteins were extracted from the cells or tissue samples using a radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Vazyme, China). The proteins were then separated using SDS-PAGE and then electro-transferred to polyvinylidene difluoride (PVDF) membranes. After soaking in 10% skimmed milk powder for 1 h, the membranes were incubated overnight at 4 °C with specific antibodies: GFAP (ABclonal, Wuhan, China), GDNF (Abcam, Cambridge, UK), S100B (Abcam, Cambridge, UK), GAPDH (Antgene, Wuhan, China), H3K9me1 (A2358, ABclonal), H3K9me3 (A2360, ABclonal), H3K27me1 (A2361, ABclonal), H3K27me3 (A2363, ABclonal), and H3 (A2348, ABclonal). Thereafter, the membranes were incubated at room temperature with HRP-labeled secondary antibodies for 1 h. The intensities of the protein bands were determined using a chemiluminescence (ECL) kit (Vazyme).