Lactate dehydrogenase a ldha

Total protein in tissues and cells was extracted and protein concentration was determined via bicinchoninic acid kits (BOSTER Biological Technology Co., Ltd., Hubei, China). The extracted proteins were conducted with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BOSTER Biological Technology) and transferred onto polyvinylidene fluoride membranes, which were blocked with 5% bovine serum albumin for 1 h. Then, the membranes were incubated with primary antibodies DACT2 (1:1000), active-β-catenin (1:5000), Glut1 (1:1000), β-actin (1:1000, all from Abcam Inc., MA, USA), p-β-catenin (1:1000), and lactate dehydrogenase A (LDH-A, 1:1000, both from Cell Signaling Technology, MA, USA) at 4 °C overnight. Next, the membranes were incubated with relative secondary antibody (Shanghai Miaotong Biotech Co., Ltd., Shanghai, China) for 1 h and developed using enhanced chemiluminescent reagent and the Bio-Rad Gel Doc EZ imager (Bio-Rad Laboratories, Hercules, CA, USA). Image J software (National Institutes of Health, Bethesda, Maryland, USA) was employed to analyze the gray values of the protein bands.

Whole cell lysates were extracted with RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland) and subjected to Western blot analysis as previously described 10. Proteins were separated on 4–15% gradient polyacrylamide–sodium dodecyl sulfate gels (Bio‐rad Laboratories, Inc., Hercules, CA), transferred to Supported Nitrocellulose membranes (Bio‐rad) using a Bio‐Rad Mini‐PROTEAN Tetracell system, followed by incubation for 1 h in a bovine serum albumin‐Tween‐20‐based blocking solution. Primary antibodies were Hexokinase I (1:1000), Hexokinase II (1:1000), phosphofructokinase (PFK) (1:1000), pyruvate kinase M1/2 isoform (PKM1/2) (1:1000), pyruvate kinase M2 isoform (PKM2) (1:1000), pyruvate dehydrogenase (PDH) (1:1000), lactate dehydrogenase A (LDHA) (1:1000), GAPDH (1:1000), and actin (1:1000), all from Cell Signaling Technology, Inc.(Danvers, MA). Blots were incubated with primary antibody overnight and then incubated for 1 h with horseradish phosphatase–conjugated anti‐rabbit or anti‐mouse secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Blots were developed using the Enhanced Chemiluminescence Kit (Pierce, Thermo Fisher Scientific, Waltham, MA) followed by autoradiography.