Fig. 5: USP25 promotes K48-linked deubiquitination of STAT3. | Cell Death & Disease

Fig. 5: USP25 promotes K48-linked deubiquitination of STAT3.

From: Ubiquitin-specific protease 25 ameliorates ulcerative colitis by regulating the degradation of phosphor-STAT3

Fig. 5

A, B Plasmids overexpressing HA-STAT3 were transfected into 293T cells. Cycloheximide was used to inhibit protein synthesis, and MG132 and chloroquine were used to inhibit ubiquitin-lysosome and autophagolysosomes, respectively, to degrade proteins. The time gradient was set to 0, 2, 4, and 8 h. Western blotting was performed to examine STAT3 protein levels corresponding to each time point. Protein ubiquitination assays were performed on NCM460 cells with low USP25 expression (C) or USP25-overexpressing NCM460 cells (E) with or without LPS treatment and the intestinal tissue of WT/Usp25−/−mice with or without DSS treatment (D). F In screening for potential lysine ubiquitination types, the deubiquitination of HA-STAT3 in response to USP25 overexpression was examined in 293T cells transfected with the wild-type and mutated Myc-Ub plasmids. G–I Ubiquitination assays determining the deubiquitination of endogenous STAT3 in NCM460 cells with low USP25 expression (G) or USP25-overexpressing NCM460 cells (H) with LPS treatment and colons of WT/ Usp25−/−mice with DSS administration. J Ubiquitination assays determining the deubiquitination of HA-STAT3-K49R/K409R/K531R/K707R/K685R in response to USP25 overexpression in 293T cells transfected with the indicated plasmids.

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