Figure 1.

Identification of PKM2 as a major celastrol-modified protein. (A) Scheme for the isolation of covalent celastrol-protein conjugates. After the incubation of macrophage proteins with celastrol-coated agarose resin, celastrol-bound proteins were eluted, separated and characterized by MALDI-TOF-MS technology. (B) WB analysis of celastrol-bound proteins. The proteins were detected by WB with anti-PKM2 antibody. CL: cell lysates; FL: flow-through fraction; W1-W4: washing fractions; E: elution fraction. (C) Validation of covalent celastrol-PKM2 conjugate. Recombinant PKM2 was in-house prepared and treated with celastrol-PEG3-biotin with or without celastrol at 4 °C overnight. The celastrol-PKM2 conjugate were examined by WB analysis using streptavidin-HRP or anti-PKM2 antibody.