Fig. 7: Gp96 blocks Trim28-mediated Eomes degradation by completion with Eomes for Trim28 binding. | Nature Communications

Fig. 7: Gp96 blocks Trim28-mediated Eomes degradation by completion with Eomes for Trim28 binding.

From: Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes

Fig. 7

A, B HEK293 cells stably expressing Eomes were transfected with siRNAs targeting indicated E3 ligases. Cells were then subjected to western blotting (A). Cells were grown for 24 h and treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses (B). C HEK293 cells stably expressing Eomes were transfected with Trim28 or a control vector. Cells were treated with 50 μg/ml CHX for the time as indicated and were then subjected to western blotting. D Flow cytometry analysis of Eomes levels in GFP- and GFP+ cell in primary NK cells infected with Trim28 GFP lentiviral vector (MOI = 10). n = 3 biologically independent samples. Mean ± SD is shown. Primary NK cells transfected with Trim28 were treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses (E), or endogenous Eomes protein was immunoprecipitated with a specific antibody for Eomes or normal rabbit IgG, followed by immunoblotting (F). G The protein-protein interaction prediction tools-ZDOCK 3.0.2 was used to predict the interaction between full-length Eomes (PDB ID: AF-O54839) and Trim28 (PDB ID: AF-Q62318). Blue represents Trim28, and the green represents Eomes. H HEK293 cells transfected with indicated plasmids were grown for 24 h and treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses. I WT and gp96 knockout HEK293 cells were transfected with His-Eomes and HA-Trim28 plasmids. Cells were grown for 24 h and treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses. J HEK293 cells stably expressing Eomes were transfected with indicated plasmids. Cells were grown for 24 h, followed by IP-Western analyses. K WT and gp96 knockout HEK293 cells were transfected with His-Eomes and HA-Trim28 plasmids. Cells were grown for 24 h, followed by IP-Western analyses. L Primary NK cells from WT and gp96 knockout mice were sorted, and endogenous Eomes protein was immunoprecipitated with a specific antibody for Eomes or normal rabbit IgG, followed by immunoblotting. M HEK293 cells stably expressing Flag-gp96 were transfected with indicated plasmids. Cells were grown for 24 h, followed by IP-Western analyses. N The protein-protein interaction prediction tools-ZDOCK 3.0.2 were used to predict the interaction between Eomes (PDB ID: AF-O54839) and Trim28 (PDB ID: AF-Q62318). Blue represents the Ring domain of Trim28, and the green represents Eomes. O The protein-protein interaction prediction tools-ZDOCK 3.0.2 were used to predict the interaction between gp96 (PDB ID: AF-P14625) and Trim28 (PDB ID: AF-Q62318). Blue represents the Ring domain of Trim28, and the green represents gp96. P A working model. The E3 ubiquitin ligase Trim28 targets Eomes for lysosomal degradation, resulting in the inhibition of NK development and function. Gp96 binds to Trim28 mainly in cytosol and Eomes mainly in nucleus, and protects Eomes from Trim28-mediated degradation. The data are representative of two independent experiments with similar results.

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