Ab115730

IHC was performed to evaluate the expression levels of Ki-67 (ab15580; Abcam), CD4 (RMA-0620, Maxim, China), CD8 (RMA-0514, Maxim, China), Glut-1 (ab115730; Abcam), PD-L1 (ab205921; Abcam), CXCL13 (ab246518; Abcam), TGF-β (ab189778; Abcam), FASN (ab99359; Abcam), CK (Kit-0009, Maxim, China), and FoxP3 (98,377, CST) following previously described procedures (Xu et al., 2021a (link); Xu et al., 2021b (link)). Opal multispectral was implemented to identify differential immune cell infiltration and PD-L1 expression in different groups on a multispectral imaging system (Vectra^® Polaris™, Shanghai, China).

Protein extraction reagent (50 mM Tris HCl, pH 7.4; 120 mM NaCl; 1% Nonidet P − 40; 0.25% deoxycholate; 0.1% sodium dodecyl sulfate) were extracted and supplemented with protease inhibitor mixture (Beyotime, Shanghai, China). The protein concentrations of the samples were then normalized using the BCA Protein Assay Kit (Pierce, Rockford, IL). Protein extracts were equally added to 10% SDS polyacrylamide gels, subjected to electrophoresis, and then transferred to nitrocellulose membranes (Amersham Bioscience). After blocking with 5% nonfat milk in PBS, related primary antibodies (anti-HIF1α (ab179483, 1:1000 dilution, Abcam, Cambridge, MA, USA), anti-HK2 (ab209847, 1:1000 dilution, Abcam, USA), anti-GLUT1 (ab115730, 1:100000 dilution, Abcam, USA) and GAPDH (ab8226, 1 µg/ml, Abcam, USA)) were incubated overnight, followed by a horseradish peroxidase conjugated secondary antibody (Santa Cruz Company). The signal was detected by a chemiluminescence substrate Kit (Millipore Company, Bedford, MA, USA).

Western blot analysis was performed as previously described. Briefly, cells were lysed in RIPA buffer to extract total proteins. Proteins were separated by SDS-PAGE gel and were electrically transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk to prevent non-specific binding of antibodies and incubated with specific primary antibodies of KCNK3 (A14745, 1:1000, ABclonal, China), GLUT1 (ab115730, 1:10000, abcam, USA), LDHA (ab47010, 1 µg/ml, abcam, USA), AMPK (ab32047, 1:2000, abcam, USA), p-AMPK (2535 T 1:1000, CST, USA) and TXNIP (ab188865, 1:1000, abcam, USA) at 4 °C overnight. Corresponding HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. Immunoreactive proteins were visualized using Image Quant LAS 4000 (GE Healthcare Life Science, Chicago, IL, USA).

Tissues and cells were lysed with a modified buffer, and western blotting was performed as described previously^{41 (link)}. The primary antibodies were as follows: MLXIPL (Abcam, ab92809), GLUT1 (Abcam, ab115730), PKM1 (Abcam, ab116271), PKM2 (Abcam, ab137852), LDHA (Abcam, ab84716), and β-actin (Abbkine, A01011). And images were captured using an Amersham Imager 600 System (GE Healthcare).

Standardized immunohistochemical stainings were performed on formalin-fixed paraffin-embedded (FFPE) xenograft tumor tissue. Five-micrometer-thick sections were placed on coated glass slides, deparaffinized, and rehydrated and then subjected to high-pressure antigen retrieval in a pressure cooker for 3 min in preheated 10 mmol/L sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 min, and nonspecific staining was eliminated by incubating the sections with 10% normal goat serum for 15 min at room temperature. The sections were incubated with the primary antibody (HIF-1α BD Bioscience 610958, GLUT1 Abcam ab115730, LDHA CST-3582, HK2 Abcam ab209847) at 4°C overnight. Sections were incubated with diluted biotinylated secondary antibody for 10 min and then incubated with the avidin-biotin-peroxidase complex for another 10 min with repeated washing steps. Staining was visualized using 3,3’-diaminobenzidine solution (Maxim, Fuzhou, China). Sections were then counterstained with hematoxylin after dehydration and scanned by Motic digital slide scanner.

After the cells were exposed to various concentrations of ZEA, the cells were collected using trypsinization. All proteins of the cells were extracted with RIPA lysis buffer. The concentration of the entire protein was detected and adjusted using a BCA protein assay kit. After the whole protein was mixed with the loading buffer and boiled for 10 min, the protein extracted was injected into a sodium dodecyl sulfate polyacrylamide gel, separated according to the size of the protein, and then the protein was transferred to a polyvinylidene fluoride film. The membrane was incubated with 5% skim milk solution; the membrane was then probed with the indicated pro-antibody at a temperature of 4 °C overnight, washed and then incubated with secondary antibody for 2 h at room temperature. Protein signals were detected using an ECL detection system. The polyclonal antibodies against GLUT1 (1:100000, ab115730), LDH (1:1000, ab101562), and MCT4 (1:1000, ab180699) were acquired from Abcam (Cambridge, MA, USA). The polyclonal antibodies against GAPDH (1:1000, 2118) and SIRT1 (1:1000, D1D7) were acquired from Cell Signaling Technology (Boston, MA, USA);

Tissue samples and cultured cells were analysed by Western blotting. Total protein was extracted using radio‐immunoprecipitation assay (RIPA) lysis buffer containing 50 mM Tris‐HCl pH 7.5, 0.25% sodium deoxycholate, 1mM EDTA, 1% NP‐40, 1 × protease inhibitor cocktail (Merck). Protein concentrations were quantified by BCA Protein Assay Kits (Thermo). Twenty micrograms of total protein was separated using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to nitrocellulose (NC) membranes. NC membranes were blocked with 5% skim milk for 1 hour at 25°C and then incubated with anti‐SLC1A3 (EAAT1) (1:1000, Abcam, ab41751), anti‐HK II (1:5000, Abcam, ab209847), anti‐GLUT1 (1:4000, Abcam, ab115730), anti‐LDHA (1:1000, Abcam, ab101562), anti‐p‐AKT (1:1000, Cell Signaling Technology, #9271), anti‐AKT (1:1000, Cell Signaling Technology, #9272) or anti‐GAPDH (1:2000, Cell Signaling Technology, #5174) overnight at 4°C. Blots were washed then incubated with secondary antibody (anti‐rabbit: HRP conjugate) (1:2000, Cell Signaling Technology, #7074) for 1 hour at 25°C. Finally, signals were detected with an enhanced chemiluminescence reagent (ECL) (Thermo) and captured using a digital imaging system (LI‐COR Bioscience).