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. 2022 Aug 15;18(14):5260–5275. doi: 10.7150/ijbs.73890

Figure 2.

Figure 2

Characterization of covalent celastrol-binding site in PKM2 and the impact on pyruvate kinase activity. (A) UPLC-MS/MS peptide mapping of covalent celastrol binding peptides from recombinant PKM2. Following overnight incubation with celastrol, recombinant PKM2 solution was resolved with SDS-PAGE, stained with Coomassie blue and characterized by UPLC-MS/MS method. (B) In silico prediction of molecular docking. (C) Formation and visualization of intracellular covalent celastrol-PKM2 conjugate. After incubating RAW264.7 cells with celastrol-PEG4-alkyne or celastrol, the conjugated celastrol-PEG4-alkyne was visualized with AFDye555-picolyl azide while the cellular PKM2 was visualized by immunofluorescence staining. The cell nuclei were detected with DAPI. Scale bar: 10 µm. (D) Inhibition of PKM2 activity by celastrol. After overnight incubation with celastrol, recombinant PKM2 protein was assayed for pyruvate kinase activity. ***p < 0.001 (celastrol vs control).