In Vitro and In Situ Assay of PKM2 Oligomers

Assay of PKM2 dimer and tetramer in vitro follows previous descriptions⁴¹ . In brief, after 40ng PKM2-FBP complex was treated with 200–300μM SNO-CoA for 10 min at room temperature, 5 μl fresh glutaraldehyde (50%) was added to a reaction mixture containing 100mM HEPES (pH 7.5) for 5 min at 37°C. The cross-linking reaction was terminated by addition of 5 μl 1M Tris-HCL (PH 8.0). Assay of PKM2 dimer and tetramer in situ was carried out as described previously^{14 (link)}. DSS (disuccinimidyl suberate; Thermo Scientific) (final 500 μM) was added to cells for 30 min at room temperature to cross-link proteins. Cells were lysed in RIPA Buffer and protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein were separated by 4–20% Criterion™ Precast Midi Protein Gel (BIO-RAD) and monomer, dimer and tetramer forms of PKM2 were detected with PKM2 antibody (sc-365684, Santa Cruz Biotechnology).