Ab101562

Cell lysates were prepared by lysing pancreatic cancer cells in RIPA lysis and extraction buffer (Thermo Fisher Scientific Inc., Waltham, MA USA) supplemented with a proteinase inhibitor cocktail (Thermo Fisher Scientific Inc.) and phosphatase inhibitor (Cell Signaling Technology, CST, Danvers, MA, USA). Equal amounts of denatured protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE; Bio‐Rad, Hercules, CA) followed by electro‐transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). Membranes were incubated with blocking buffer containing 5% bovine serum albumin (BSA; Sigma‐Aldrich) for 2 hours before overnight incubation with the relevant antibodies: LDHA (1: 1000, ab101562, Abcam), AMPKα (D5A2) (1:1000, CST), Phospho‐AMPKα (Thr172) (40H9) (1:1000, CST), mTOR (7C10) (1:1000, CST), Phospho‐mTOR (Ser2448) (D9C2) (1:1000, CST), β‐Actin (D6A8) (1: 1000, CST). The detailed reactivity of each antibody used are included in the supplementary materials (Table S1). HRP‐conjugated secondary antibody (1: 5000, CST) was applied to the membrane for 2 hours at room temperature, and were then visualized using the Amersham™ ECL Select™ western blotting detection reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK) using a chemiluminescence imaging system (Bio‐Rad, Hercules, CA, USA).

Total proteins were extracted from cells or tissues using RIPA lysis buffer (Promega, Madison, WI, USA). Proteins were separated on an 8% SDS-PAGE gel and then transferred to PVDF membranes. The membrane was blocked with 5% skim milk at room temperature for 1 h and then incubated with primary antibodies (anti-DLAT, 1:1000 dilution, Abcam, ab51608; anti-SP1, ab101562, 1:1000 dilution, Abcam) at 4 °C overnight, followed by incubating with horseradish peroxidase (HRP) conjugated β-actin secondary antibodies (1:5000) at room temperature for 90 min. The protein bands were scanned and visualized by the GS700 imaging densitometer (Bio-Rad Laboratories) and analyzed by Image Studio software.

NSCLC tissue or cellular proteins were respectively extracted with cell lysis buffer (Promega, Madison, WI, USA). Thirty micrograms of proteinextraction was separated on an 8% SDS-PAGE gel and then transferred to nitrocellulose membranes. After blockage by milk, the membranes were incubated for 2 h at room temperature with primary antibody (anti-HIF1A, 1:1000 dilution, Abcam, ab51608; anti-LDHA, ab101562, 1:1000 dilution, Abcam). Then, blots were incubated at room temperature for 90 min with horseradish peroxidase (HRP) conjugated beta-actin secondary antibodies (diluted 1:5000). The bands were scanned and visualized by GS700 imaging densitometer (Bio-Rad Laboratories) and analyzed by Image Studio software.

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);

The radioimmunoprecipitation assay (RIPA) buffer was used to extract total protein, supplemented with 1% protease inhibitors (P8340, Sigma-Aldrich) and phosphatase inhibitors (P5726, Sigma-Aldrich). The bicinchoninic acid (BCA) assay was used to measure protein concentration. Western blot analysis was performed, as previously described (12 (link)). THBS2 (sc-136238, Santa Cruz Biotechnology), TLR4 (ab30667, Abcam), Ki67 (Proteintech Group, Inc.), HIF-1α (ab2185, Abcam), GLUT1 (ab115730, Abcam), HK2 (ab104836, Abcam), ALDOA (ab150396, Abcam), PKM2 (ab137852, Abcam), and LDHA (ab101562, Abcam) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H+L) and HRP-conjugated AffiniPure goat anti-mouse IgG (H+L) were obtained from Proteintech Group, Inc. (Jackson).

After transfection for 48 h, NSCLC cells were harvested. The cell lysates were obtained using the Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). Cell lysates (30 μg) were subjected to 12% separating gel and then blotted to the polyvinylidene fluoride (PVDF) membrane (Bio-Rad). After blocking, the PVDF membrane was mixed with the primary antibodies, including anti-hexokinase 2 (anti-HK2, ab209847, Abcam, Cambridge, MA, United States), anti-lactate dehydrogenase A (anti-LDHA, ab101562, Abcam), anti-KRAS (ab180772, Abcam), anti-CD9 (ab92726, Abcam), anti-CD81 (ab79559, Abcam), anti-tumor susceptibility 101 (anti-TSG101, ab125011, Abcam), anti-Golgi matrix protein 130 kDa (anti-GM130, ab32337, Abcam), and anti-β-actin (anti-20272, Abcam). After washing three times using PBS-Tween 20 (PBST), horseradish peroxidase(HRP)-combined secondary antibody (ab205718, Abcam) was utilized to incubate with the membrane for 2 h at room temperature. After washing with PBST, protein signals were visualized using the enhanced chemiluminescent (ECL) system (Beyotime).