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. Author manuscript; available in PMC: 2013 Jul 15.
Published in final edited form as: Nat Chem Biol. 2012 Oct;8(10):839–847. doi: 10.1038/nchembio.1060

Figure 3. Activators promote PKM2 tetramer formation and prevent inhibition by pTyr signaling.

Figure 3

(a) Sucrose gradient ultracentrifugation profiles of purified recombinant PKM2 and effects of FBP and TEPP-46 on PKM2 subunit stoichiometry. Recombinant PKM2 was transiently exposed to FBP prior to addition of TEPP-46. After centrifugation, fractions were collected, analyzed by SDS-PAGE and stained with Coomassie Blue. Relative protein amounts were calculated by band densitometry on a LiCOR Odyssey infrared imaging system. (b) A549 cells were treated with 100 μM pervanadate for 10 min. in the presence or absence of TEPP-46, lysed hypotonically, and were analyzed by size exclusion chromatography. Chromatographic fractions were then subjected to western blotting with a pyruvate kinase antibody to assess the stoichiometry of PKM2 subunit association under these conditions. Uncropped blots are shown in Supplementary Fig. 10. (c) Pyruvate kinase activity assays in A549 cells treated with pervanadate as in (b) in the presence of DMSO, 1 μM TEPP-46 or 1 μM DASA-58 (N=3, p=0.0044 by 2-way ANOVA).