A0216

Cells were lysed in RIPA buffer (Beyotime, China) with 1% PMSF (Biosharp, China). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Epizyme, China) and transferred onto PVDF membrane (Millipore, USA). Primary antibodies were applied at 4 °C overnight and HRP-conjugated secondary antibodies were applied for an hour at room temperature. The immunocomplexes were detected with ECL Western Blotting Substrate (NCM Biotech, China), visualized with Tanon (5200multi 4600SF, Tanon, USA). GAPDH was used as the internal control. Primary Antibodies included rabbit antiMAML3 (1:500, Biorbyt, UK), mouse antiGAPDH (1:20000, Beyotime, China). Secondary antibodies (A0208 and A0216, Beyotime, China) were diluted in 1:1000.

Total cells collected from different experiments were obtained by lysing in RIPA buffer. The protein concentration was determined using the BCA Protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Cell lysates were subjected to SDS-PAGE electrophoresis and proteins were blotted onto PVDF membranes. The membranes were further incubated with primary antibodies PDK1 (1:1500, ab110025, Abcam Inc, Cambridge, MA, USA) and GAPDH (1:2000, AF0006, Beyotime Biotechnology, Shanghai, China) followed by incubation with an HRP‑conjugated secondary antibody (1:3000, A0216, Beyotime Biotechnology, Shanghai, China). Exposure was performed using enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA).

Western blot analyses were performed as previously described^{65 (link),66 (link)}. Primary antibodies were listed as follows: anti-VDAC2 (ab47104, Abcam); anti-PFKP (ab204131, Abcam); anti-β-actin (mAb#5125, Cell Signaling Technology, Danvers, MA); anti-COX IV (#4850, Cell Signaling Technology); anti-OLIG2 (ab109186, Abcam, Cambridge, UK); anti-β-tubulin (ab179513, Abcam); anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany); and anti-SOX2 (#3579, Cell Signaling Technology). Secondary antibodies are horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) (A0208, Beyotime, Shanghai, China) and HRP-labeled goat anti-mouse IgG (H + L) (A0216, Beyotime). Immuno-bands were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).

A BCA Protein Assay Kit (23225, Thermo Scientific Pierce, IL, USA) was used for protein quantification. Rabbit monoclonal antibody anti‐EGR1 (1:1000; 4153, Cell Signalling Technology, MA, USA), rabbit polyclonal antibody anti‐Exph1 (1:500; orb340819, Biobyt, CA, USA), mouse monoclonal antibody anti‐IL10 (60269‐1‐Ig, proteintech, IL, USA), rabbit polyclonal antibody anti‐β‐actin (1:2000, NC021, Zhuangzhibio, Shaanxi, China), horseradish peroxidase‐conjugated secondary antibody (A0216, Beyotime Biotechnology, Shanghai, China) and enhanced chemiluminescence detection (32132, Thermo Scientific Pierce, IL, USA) were employed.

Whole protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime) and quantified using the bicinchoninic acid Protein Assay Kit (Beyotime). Briefly, a 20 μg protein aliquot was loaded into 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for separation, followed by electroblotting on a polyvinylidene fluoride membrane (Merck Millipore, Burlington, MA, USA), blocking using 5% fat-free milk, and overnight incubation with the following primary antibodies: anti-UBR5 (ab70311; Abcam), anti-AXIN1 (ab55906; Abcam), anti-β-catenin (ab32572; Abcam), anti-Survivin (ab76424; Abcam), anti-C-myc (ab39688; Abcam), anti-lactate dehydrogenase A (LDHA, ab101562; Abcam), anti-pyruvate kinase isozymes M2 (PKM2, ab137852; Abcam), anti-H3 (ab1791; Abcam), and anti-GAPDH antibody (60004-1-1G; ProteinTech, Rosemont, IL, USA). They were washed thrice with Tris-buffered saline with Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies (A0208, A0216; Beyotime). Finally, the blot was imaged using the Enhanced Chemiluminescence Detection Kit (Pierce Biotechnology), and the band intensity was measured using Image-Pro Plus 6.0 software, with GAPDH as the loading control.

Cells were lysed in RIPA Lysis Buffer (Beyotime) and mixed with PMSF (Beyotime), and protein concentrations of the resulting lysates were quantified using a BCA Protein Assay Kit (Beyotime). A total of 30 μg of protein from each sample was separated using SDS-PAGE on a 10% gel and transferred to a polyvinylidene fluoride membrane. The membranes were probed with the following antibodies: 1:1000 rabbit polyclonal anti-CHERP antibody (ab15951, Abcam); 1:1000 rabbit polyclonal anti-cleaved caspase-3 antibody (AC033, Beyotime); mTOR substrate antibodies (9862, CST); 1:800 polyclonal anti-DR5/TNFRSF10B antibody (BS60081, Bioworld); 1:1000 polyclonal anti-Bip antibody (3177, CST); 1:1000 CHOP polyclonal antibody (2895, CST); 1:500 AKT polyclonal antibody (4691, CST); 1:1000 polyclonal anti-P-Akt (Ser473) antibody (4060, CST); 1:800 polyclonal anti-cleaved caspase-8 antibody (E1A5267, EnoGene); and 1:800 polyclonal anti-ATF4 antibody (E2A6008, EnoGene). For the cell cycle protein assay, either a cell cycle regulation antibody sampler kit (9932, CST) or a 1:2000 α-tubulin mouse polyclonal antibody (AT819, Beyotime) was used. The secondary antibodies utilized were horseradish peroxidase-conjugated goat anti-mouse (A0216, Beyotime) and goat anti-rabbit IgG (A0208, Beyotime).