Fig. 2

Identification of aldolase A, α-enolase and PKM2 as the binding partners of DA in MN9D cells. A ABPP workflow for the identification of the DA protein targets. Live cells were incubated with DA-probe (blue circle). For fluorescence imaging, cells were fixed and permeabilized, followed by the attachment of TAMRA-azide tag (red circle) by CuAAC reaction; otherwise, cells were lysed and the DA-P labeled proteome were ligated by CuAAC to TAMRA-azide for fluorescence SDS-PAGE analysis, or to biotin azide (purple circle) for LC–MS/MS based protein targets identification. B Volcano plot representing proteins labeled by DA-P (100 μM) for 3 h in live cells versus samples pre-treated with 800 μM DA for 4 h followed by co-treatment with DA-P (100 μM) for 3 h (fold change ≥ 2 and p value < 0.05). Red: upregulated genes. C In situ pull-down to verify the interaction between DA and aldolase A, α-enolase and PKM2 proteins. Cells treated with 100 μM DA-P or with an excess of the DA (800 μM, 4 h) plus the DA-P for 3 h were lysed, followed by the attachment of biotin-azide by CuAAC reaction. D CETSA to verify DA binding to α-enolase. Cell lysates incubated with 100 μM DA for 2 h were subjected to CESTA-western blot analysis. E Representative fluorescence images of intracellular aldolase A or α-enolase in MN9D cells incubated with 100 μM DA-P for 3 h. Error bars, mean ± s.d. Scale bar, 10 μm. *, p < 0.05; **, p < 0.01