Anti hk2

Harvested cells were lysed by RIPA buffer. After sonication, samples were centrifuged at 12 000 g for 15 min at 4°C. Total protein concentration was determined by applying DC Protein Assay Kit I (Bio‐Rad, USA). Proteins were transferred to Hybond nitrocellulose membranes (USA) after separation on 12% SDS‐PAGE. 5% skim milk was added to seal the membrane in Tris‐buffered saline (pH 7.5) at room temperature. The membrane was incubated with the primary antibody overnight at 4°C, followed by 4‐h incubation with the secondary antibody at room temperature. Protein bands were developed by ECL kit (Millipore, USA). Protein levels were assessed by ImageJ (USA). Primary antibodies including anti‐CD133, anti‐CD44, anti‐Oct‐4, anti‐HK2, anti‐PKM2, anti‐LDHA, anti‐β‐actin, and secondary antibody anti‐IgG were purchased from Abcam (UK).¹⁶

The cell lysates were mixed with 4 × Laemmli sample buffer (Bio-Rad) and heated at 95 °C for 5 min. The proteins were separated using SDS-PAGE (7.5%–10% gels) and transferred to nitrocellulose membranes (GE Healthcare). After blocking, the membranes were incubated with primary antibodies overnight at 4 °C and HRP-conjugated secondary antibodies for 1 h. Proteins were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgGs using an enhanced chemiluminescence detection kit (Immunostar LD; WAKO; Osaka, Japan). Anti-GPR81 (#NLS2095, 1:1000) was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies against MCT1 (#sc-365501, 1:100) and PFK1 (#sc-166722, 1:100) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-MCT4 (#22787-1-AP, 1:10,000) was purchased from Proteintech. Anti-HK2 (#ab104836, 1:1000) was purchased from Abcam (Cambridge, England). Anti-LDHA (#2012, 1:1000) was purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-β-actin (M177-3) was purchased from MBL (Nagoya, Japan). Uncropped images of western blots are shown in Supplementary Fig. S6 online.

Proteins were extracted with RIPA lysis buffer with mixed protease inhibitors (Sigma, United States). Proteins (30 μg) were separated on SDS-PAGE gel, followed by transfer to nitrocellulose membrane (MilliporeSigma, United States). The membranes were then blocked with 5% skim milk and incubated at 4°C overnight with the primary antibody: anti-STK35 (ab136695; Abcam, United States), anti-NEDD4L (#2740; Cell Signaling Technology, United States), anti-cleaved caspase-3 (ab2302; Abcam, Cambridge, MA, United States), anti-cleaved caspase-9 (ab2324; Abcam, United States), anti-GLUT1 (ab40084; Abcam, United States), anti-HK-2 (ab209847; Abcam, United States), anti-AKT (#9272; Cell Signaling Technology, United States), anti-p-AKT (#4060; Cell Signaling Technology, United States), or β-actin (#3700; Cell Signaling Technology, United States). Following primary antibody incubation, the membranes were incubated with HRP-conjugated secondary antibody (Beyotime, China). The membranes were visualized by using ChemiDoc Imaging Systems (Bio-Rad, United States).