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. 2022 Sep 5;23(17):10161. doi: 10.3390/ijms231710161

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© 2022 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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TRAF4 regulates SETDB1 stabilization and mediates atypical ubiquitination of SETDB1. (A) TRAF4 and SETDB1 mRNA levels were examined by qRT-PCR in glioblastoma cells. (B,C) Control group and TRAF4 knockdown cells were treated with CHX in a time gradient (0 h, 1 h, 2 h, 4 h). The protein expression of SETDB1 was detected by Western blot. The grey values were calculated by ImageJ. (D,E) MYC-tagged SETDB1, HA-tagged Ub, and (D) shTRAF4/ (E) Flag-tagged TRAF4 constructs were cotransfected into 293FT cells. The ubiquitinated SETDB1 proteins were pulled down with anti-MYC antibody and immunoblotted with anti-HA antibody. (F) The Flag-tagged TRAF4, MYC-tagged SETDB1, HA-tagged Ub, and ubiquitin mutant constructs were cotransfected into 293FT cells. The 293FT cells were treated with MG132 for ubiquitination assays. Student’s t test was performed to analyze significance; ** p < 0.01, NS: not significant.