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. 2019 Feb 11;129(3):1129–1151. doi: 10.1172/JCI121987

Figure 5. Characterization of triptolide as a MEX3C inhibitor.

Figure 5

(A) Top panel: flow chart of sandwich ELISA using His6-TUBE and Ub-PTEN (K80) antibody screening customized compound library and the generation of PTENK27-polyUb was detected by OD450. Bottom panel: The log2 of relative fold change of polyUb chain formation activity was shown. (B) Illustration of Bio-Linker and Bio-TPL. (C) Streptavidin pulldown using Bio-Linker or Bio-TPL and Col4a3+/+ or Col4a3–/– kidneys. The Bio-TPL–binding proteins were excised (blue box) and subjected to LC-MS analysis. (D) Protein scores of protein identification of LC-MS. (E) Competition curve determination of KD value of TPL and indicated recombinant proteins with 2-serial fold dilution. Error bars indicate SD; n = 3 independent experiments. (F) Competition curve determination of IC50 value of TPL on the enzymatic activity of indicated E3 ligases. The IC50 values of TPL against each E3 ligase are shown. Error bars indicate SD; n = 3 independent experiments. (G) In vitro ubiquitination assay using indicated recombinant proteins with or without TPL followed by immunoblotting detection (ERCC3/MEX3C molar ratio 10:1). (H–I) Streptavidin pulldown using Bio-TPL and indicated recombinant proteins of MEX3C followed by immunoblotting detection using anti-FLAG antibody.