Anti total h3 primary antibody

Protein was extracted from cells using RIPA buffer (89900, Thermo Fisher Scientific). A total of 60 µg of protein (from whole cell extract) was separated by electrophoresis in a 4–15% precast protein gel (4561086, BioRad) and transferred to PVDF membranes. Blocking was subsequently performed with 5% non-fat milk in TBST, followed by incubation with anti-H3K27Ac antibody at 1:500 dilution (8173S, Cell Signaling Technology) overnight. After 5 washes with TBST, membranes were incubated with HRP-conjugated anti-Rabbit IgG antibody at 1:1000 (7074 Cell Signaling Technology) for 1 h. Pierce ECL Plus (32132, Thermo Fisher Scientific) was used to detect protein bands. Blots were then stripped (46430, Thermo Fisher Scientific) and re-probed with anti-total H3 primary antibody at 1:1000 dilution (14269S, Cell Signaling Technology) as a loading control. HRP-conjugated anti-Mouse IgG antibody (7076, Cell Signaling Technology) was used to detect total H3 signal. Densitometry analysis was performed with image J.

Frozen tissue was thawed and dissociated with gentleMACS Dissociator (130-093-235, Macs Miltenyi Biotec). Protein was extracted in Tissue Extraction Reagent (FNN071, Thermo Fisher Scientific) and concentration determined with Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific). 60 μg of protein was separated by electrophoresis in a 4–15% precast protein gel (4561086, BioRad) and transferred to PVDF membranes. Blocking was subsequently performed with 5% non-fat milk in TBST, followed by incubation with anti-H3K27Ac antibody at 1:500 dilution (8173S, Cell Signaling Technology) overnight. After 5 washes with TBST, membranes were incubated with HRP-conjugated anti-Rabbit IgG antibody at 1:1000 (7074 Cell Signaling Technology) for 1 hour. Pierce ECL Plus (32132, Thermo Fisher Scientific) was used to detect protein bands. Blots were then stripped (46430, Thermo Fisher Scientific) and re-probed with anti-total H3 primary antibody at 1:1000 dilution (14269S, Cell Signaling Technology) as a loading control. HRP-conjugated anti-Mouse IgG antibody (7076, Cell Signaling Technology) was used to detect total H3 signal. Protein from a patient-derived DIPG cell line SF8628 was extracted with RIPA buffer (89900, Thermo Fisher Scientific) and processed in parallel with tissue specimens as a positive control. Densitometry analysis was performed with image J.

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.