Glucose-Dependent PKM2 Expression in 661W Cells

The 661W cells were plated in 6-well plates overnight in DMEM with 10% FBS. Subsequently, media were replaced with DMEM, no glucose (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. 11966025), supplemented with either 5.5 mM or 25 mM glucose and 10% dialyzed serum. Forty-eight hours later, the cells were harvested and lysed in RIPA Lysis and Extraction Buffer containing protease and phosphatase inhibitors (Cell Signaling, Cat No. 5872). The protein concentration was estimated using the Pierce BCA protein assay kit. Twenty-five micrograms of protein was diluted in Laemmli buffer (Bio-Rad, Hercules, CA, USA, Cat No. 1610747) complemented with β-mercaptoethanol (Millipore-Sigma, Burlington, MA, USA, Cat No. M6250) and run on a 4–20% Mini-PROTEAN^® TGX™ Precast Gel (Bio-Rad, Hercules, CA, USA, Cat No. 4561093EDU). The blots were processed and developed as we have previously described [47 (link)]. The antibodies that were utilized were as follows: PKM2-specific monoclonal antibody (Proteintech, Rosemont, IL, USA, Cat No. 60268-1-lg) and anti-α-tubulin antibody, mouse monoclonal (Sigma-Aldrich, St. Louis, MO, USA, Cat No. T6199).