Gadd45b

Total proteins from TBP/Q₇₉-GFP SH-SY5Y cells were obtained using a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and a protease inhibitor cocktail (Sigma-Aldrich). After quantitation using a protein assay kit (Bio-Rad, Hercules, CA, USA), proteins (20 μg) were separated on 10−12% SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich) by reverse electrophoresis. After blocking, the membrane was probed with CREB (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), pCREB (S133) (1 : 1000; Santa Cruz Biotechnology), BCL2 (1 : 500; BioVision, Milpitas, CA, USA), GADD45B (1 : 1000; Abcam, Cambridge, MA, USA), BAX (1 : 500; BioVision), NRF2 (1 : 500; Santa Cruz Biotechnology), AMPKα (1 : 1000; Cell Signaling, Danvers, MA, USA), pAMPKα (T172) (1 : 1000; Cell Signaling), GAPDH (1 : 1000) (MDBio Inc., Taipei, Taiwan), or β-actin (1 : 5000; Millipore, Billerica, MA, USA) primary antibody at 4°C overnight. The immune complexes were detected using a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1 : 10000; GeneTex, Irvive, CA, USA) and a chemiluminescent substrate (Millipore).

Both procedures were carried out as previously described.²¹ In brief, samples were fixated in 4% paraformaldehyde and consequently paraffin‐coated. Thin tissue segments (4 μm) were prepared, and H&E staining was performed. For IHC, following heat antigen retrieval, the tumor segments were incubated with primary antibodies at a recommended dilution concentration, including Ki67 (Abcam, ab16667), PCNA (Servicebio, GB11010), Cleaved caspase 3 (CST, #9664), LSD1 (CST, #2139), H3K4me1 (CST, #5326), H3K4me2 (CST, #9725), and GADD45B (Abcam, ab105060). Consequently, the slides were incubated with biotinylated secondary antibody, visualized with 3,3′‐diamino‐benzidine substrate, and photographed using an optical microscope (Olympus). Relative protein expression assessment was attained through the integral optical density (IOD) and area, collected by Image J software, together with average optical density (AOD) calculation. The AOD reflected targeted proteomic expression per unit area.