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. 2021 Sep 13;39(9):1262–1278.e7. doi: 10.1016/j.ccell.2021.07.003

Figure 7.

Figure 7

Identification of domains critical for TRIM8-mediated degradation of EWS/FLI

(A) Schematics of EWS/FLI deletion mutants.

(B) EWS/FLI, N-EWS, and C-FLI mutants tagged with 3XFLAG were co-transfected with either TRIM8 or a vector control in 293T cells. After 48 h, cells were lysed and immunoblotted with the indicated antibodies.

(C) 293T cells were co-transfected with TRIM8 and EWS/FLI, N-EWS, C-FLI, or a vector control for 48 h, treated with 500 nM carfilzomib for 6 h, lysed, immunoprecipitated, and immunoblotted with the indicated antibodies.

(D) 293T cells transfected with the indicated constructs for 48 h, treated with 500 nM carfilzomib for 6 h, lysed, immunoprecipitated, and immunoblotted with the indicated antibodies.

(E) Schematic of TRIM8 structural domains and mutants.

(F and G) 293T cells transfected with the indicated constructs for 48 h, treated with 500 nM carfilzomib for 6 h, lysed with either lysis buffer (F) or ubiquitination lysis buffer (G), immunoprecipitated, and immunoblotted with the indicated antibodies. Data representative of three independent experiments.

(H) Schematic depicting fusion oncoprotein-specific regulators as potential therapeutic targets. EWS/FLI-specific regulation of TRIM8 is shown as an example.