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. Author manuscript; available in PMC: 2024 Feb 16.
Published in final edited form as: Cell Chem Biol. 2023 Feb 6;30(2):203–213.e17. doi: 10.1016/j.chembiol.2023.01.004

Figure 3. Identification of CDK9 degrading E3 ligase(s) recruited by 955.

Figure 3.

(A) Schematic illustration of the TurboID-bait assay to identify CDK9 degrading E3 ligase(s) recruited by 955; (B) The biotinylated proteins identified by MS in 293T cells. E3 ligases are labelled with KEAP1 highlighted in red; (C) Western blot analysis to validate KEAP1 recruitment by 955. Representative immunoblots from two independent experiments are shown. (D) KEAP1 inhibition with CDDO-ME and CDDO-IM blocks 955-induced CDK9 degradation in MOLT4 cells; (E) Inhibition of KEAP1 with DMF blocks 955-induced CDK9 degradation in MOLT4 cells. (F) KEAP1 knockout blocks 955-induced CDK9 degradation. KEAP1 depleted (sg1 and sg2) and control (sgNC) H1299 cells were either untreated or treated with 955 for 16 h. (G) Ectopic expression of Halo-tagged KEAP1 in KEAP1 stable knockout cells rescues 955-induced CDK9 degradation. KEAP1-sg1 stable knockout H1299 cells were transfected with or without Halo-KEAP1 for 48 h and then the cells were reseeded and treated with 955 for 6 h. Representative immunoblots are shown and β-actin was used as a loading control in all immunoblot analyses. In panels D-G, representative immunoblots are shown and β-actin was used as an equal loading control. The quantification of the relative CDK9 protein content in the immunoblots is presented as mean ± SD (n = 2 biologically independent experiments) in the bar graph (bottom panel). Statistical significance was calculated with unpaired two-tailed Student’s t-tests. *P < 0.05; **P < 0.01; NS: not significant. (H) Gel-based ABPP assay demonstrates that 955 competes for IA-Alkyne binding to KEAP1 in a dose-dependent manner. Representative immunoblots from two independent experiments are shown. The quantification of the relative IAyne-labeled His-KEAP1 protein content is presented as mean ± SD (n = 2) in the bar graph (bottom panel). Statistical significance was calculated with unpaired two-tailed Student’s t-tests. *P < 0.05. (I) nanoBRET assay demonstrates that 955, but not 336, can induce the formation of the intracellular ternary complexes in live 293T cells. Data are expressed as mean ± SD of three biological replicates. See also Figure S4 and Table S2.