Ab101562

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).

The level of total protein in the supernatant of the NSCLC cell lysis was determined by the BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific, China). The samples were boiled for 10 min at 95°C. An aliquot of 30 mg of protein was separated using 10% SDS–PAGE gel, followed by PVDF membrane transfer, blocking for 1 h in 5% skim milk, and incubation with separate antibodies (ABCAm, Inc., USA)—1:1000 diluted antibody Ab127548 (anti-RNF180), 1:500 diluted antibody Ab39688 (anti-c-Myc), 1:5000 diluted antibody Ab227198 (anti-HK-2), 1:1000 diluted Ab101562 antibody (anti-LDHA), and 1:2000 diluted anti-GAPDH antibody (#5174, Cell Signaling Technology)—overnight at 4°C. Thereafter, the membranes were incubated with horseradish peroxidase secondary antibodies (A0208 (1:200), A0181(1:200), and A0216(1:200); Beyotime Biotechnology) at room temperature for 1 h. The ECL plus substrate (GE Healthcare, USA) with the LAS-400 Image Analyzer (FujiFilm Medical Systems, USA) was used for the detection of the horseradish peroxidase signal.