Bicinchoninic acid protein assay

Cells were harvested and whole-cell lysates were prepared using PRO-PREP protein extraction solution (iNtRON Biotechnology, Gyeonggi-do, Korea). To separate the nuclear and cytoplasmic fractions from the total cell lysate, a nucleus/cytosol fractionation kit (BioVision, Milpitas, CA, USA) was used in accordance with the manufacturer’s instructions. Protein concentration of samples was determined by the bicinchoninic acid protein assay (BioRad, Hercules, CA, USA). Protein samples were treated at 55°C for 10min in 2% SDS solution containing 5% β-mercaptoethanol, separated in 10% SDS-polyacrylamide gels, and transferred onto nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing 0.05% Tween 20 and 5% non-fat dried milk, and probed overnight at 4°C with primary antibodies. Immunoblots were washed with TBS containing 0.05% Tween 20 and 1% non-fat milk, and incubated with secondary antibodies conjugated with horseradish peroxidase against mouse IgG or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Immunoreactive proteins were visualized using the ECL detection system (Pierce, Rockford, IL, USA). Each western blot analysis was performed in triplicate.

After treatment, cells were rinsed twice using chilled PBS and then lysed and collected using a radioimmunoprecipitation assay buffer (Thermo) plus protease and phosphatase inhibitor cocktails (Thermo). After sonication, cell lysate was centrifuged at 15 000 × g for 30 minutes at 4°C and supernatant was collected in an eppendorf tube and stored at −80°C. Protein concentration was assessed using a bicinchoninic acid protein assay (Bio‐Rad) as per the manufacturer's instructions. Fifteen to thirty μg of protein was resolved using SDS‐PAGE, followed by electro‐transferred onto a methanol‐soaked polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% milk or 2% bovine serum albumin (for phosphorylated protein) in Tris‐Buffered Saline‐Tween 20 (TBST) and then incubated with primary antibody overnight at 4°C. The membrane was then rinsed with TBST and the immunocomplex on membrane was detected using horse‐radish peroxidase‐conjugated secondary antibody, and the final immunocomplexes were visualized using fluorography with an enhanced chemiluminescence reagent (GE Healthcare Life Sciences).

Immunoblotting was performed as described previously [10] (link). Briefly, cell pellets were snap frozen and then lysed on ice for 1 h in buffer containing 1% Triton X100, 0.1% SDS, protease inhibitor cocktail (BDBioscience) and phosphatase inhibitors I and III (Sigma). Lysates were pre-cleared by centrifugation and protein content determined by bicinchoninic acid protein assay (Biorad). Proteins were separated on 10–20% SDS-PAGE gradient gels (Biorad). Blots were probed for hexokinase I (Millipore, MAB1532), hexokinase II (Abcam, ab3279), hexokinase III (Abcam, ab126217), pyruvate kinase M2 (Cell Signaling Technology, 3198), cytochrome c (BDBiosciences, 556433), small ribosomal subunit S6 (Cell Signaling Technology, 2217), phospho-Ser235/6 small ribosomal subunit S6 (Cell Signaling Technology, 2211), Glut1 (Abcam, ab115730), mitochondrial complexes II-V (Abcam, ab110413), followed by mouse- or rabbit-conjugated horseradish peroxidase (HRP) (Cell Signaling Technology). HRP-conjugated antibodies were detected by enhanced chemiluminescence detection (Thermofisher).

Cells were cultured in growth media, then lysed in proteome profiling buffer (50 mM HEPES-NaOH, 150 mM NaCl, 0.5% (v/v) Triton X- 100, 1 mM EDTA, 1 mM EGTA, 10 μg/mL aprotinin,10μg/mL leupeptin, 1 mM PMSF, 10 mM NaF, 50 ng/mL calyculin A, 1% (v/v) phosphatase inhibitor mixture 2, 2.5 mM Na₃VO₄, pH 7.5). Total protein measurements were determined using the Bicinchoninic acid protein assay (Bio-Rad). About 100 µg of protein extracts were denatured with 6 M urea in 25 mM ammonium bicarbonate, before reduction with 5 mM TCEP at 37 °C for 1 h and alkylation with 32 mM iodoacetamide in the dark for 1 h. Alkylation was stopped by addition of 27 mM DTT. The samples were then diluted 1:10 with ammonium bicarbonate and digested with a 1:50 ratio of modified trypsin (Promega) to protein weight at 37 °C for 18 h. Tryptic digests were slightly acidified with 10% (v/v) TFA to pH 2–3, desalted with a C18 spin column (ThermoFisher Scientific), and eluted with 0.1% (v/v) TFA/40% (v/v) acetonitrile. Peptides were dried with a speed vacuum and resuspended in 2% (v/v) acetonitrile/0.1% (v/v) FA prior to mass spectrometry analysis.