Hrp 60004

15 µL protein sample was loaded into 10% polyacrylamide gel. Electrophoresis and transfer were performed using Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, USA). The membrane was blocked for 1 h with 5% BSA in TBST buffer (Tris: 20 mM, NaCl: 150 mM, Tween® 20 detergent: 0.1% (w/v)). Immunoblot analysis was conducted with the following antibodies: β-Actin (Sigma, A2228, 1:1000); H3K27Me3 (Abcam, ab6002, 1:1000); Histone 3 (Abcam, ab1791, 1:1000); anti-Brg1 (Abcam, ab4081, 1:1000); Ezh2 (Cell Signaling, D2C9 #5246, 1:1000); REST/NRSF (Millipore, 17-641, 1:1000); GAPDH‐HRP (Prosci, HRP‐60004, 1:1000); goat anti‐mouse IgG HRP (ThermoFisher Scientific, 62‐6520, 1:2500), and goat anti‐rabbit IgG HRP (ThermoFisher Scientific, 65‐6120, 1:2500). Protein bands were developed with Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific, 32209) and were imaged using the ChemiDoc MP Imaging system (Bio‐Rad, Hercules, CA, USA). Relative band densities were analyzed using ImageJ.

Immunoblots were separated by SDS-PAGE and transferred to a PVDF membrane for analysis. All blots were blocked in 5% BSA (Gold Biotechnology) in 1X TBST (American Bio), and then were probed overnight at 4 °C, with primary antibodies raised against: PPM1D (SCBT F-10 sc-376257, 1:1000), GAPDH (Proteintech group HRP-60004, 1:5000), Actin (ThermoFisher MA5-11869, 1:2000), γH2AX pS139 (CST 2577, 1:1000), NAPRT (Proteintech group 66159-1-Ig, 1:2000), NAMPT (CST 86634, 1:1000), pCHK2 T68 (CST 2197, 1:1000), H3K27M (CST 74829, 1:1000), or p53 (CST 9282, 1:1000). Blots were then washed with 1X TBST and incubated with HRP conjugated- anti-mouse (ThermoFisher 31432, 1:10,000) or anti-rabbit (ThermoFisher 31462, 1:10,000) secondary antibodies for 1 h at room temperature (RT). Immunoblot exposure was carried out using Clarity Western ECL substrate (BioRad), and imaged on a ChemiDoc (BioRad) imaging system. Uncropped and unprocessed scans of all western blots shown are available in the Source Data file.