GSK-J4 Effects on H3K27me3 in Hypothalamic Neurons

2 DIV hypothalamic neurons were treated with 0.5, 1, 1.8 µM GSK-J4 (Sigma-Aldrich) or vehicle for 24 h and then washed, harvested at 4 °C in RIPA buffer with protease and phosphatase inhibitors, and proteins were resolved and transferred onto nitrocellulose membranes (GE Healthcare, UK) as previously described [44 (link)]. Membranes were blocked 90 min at room temperature (RT) in Tris-buffered saline containing 0.1% Tween 20 and 5% BSA, and then incubated overnight at 4 °C with 1:1000 anti-H3K27me3 primary antibody (Cell Signaling Technology, USA). After that, membranes were incubated 1 h at RT with 1:10000 infrared dye-conjugated secondary antibody (LI-COR, USA) and proteins were visualized by Odyssey Infrared Imaging System (LI-COR). After H3K27me3 visualization, blots were stripped and then re-probed with 1:2000 anti-total H3 primary antibody (Cell Signaling Technology) to ensure equal protein loading. Densitometric analyses were performed with the ImageJ software (National Institutes of Health; freely available at https://imagej.nih.gov). Data are presented as a ratio of H3K27me3/total H3 of 3–4 independent cultures.