Fig. 5: TRIB3 interacts with MYC to inhibit the UBE3B:MYC axis.

a In vitro ubiquitination assays show that the TRIB3 or TRIB3-KDC protein decreased polyubiquitination of MYC mediated by UBE3B and UBCH3. b The TRIB3 protein or the KDC domain of TRIB3 decreased the interaction of MYC and UBE3B in the purified system. c Neither the TRIB3 protein nor the KDC domain of TRIB3 affected the interaction of UHCH3 and UBE3B in the purified system. d The interaction of TRIB3 and MYC in Raji cells was evaluated by Co-IP assays. e Colocalization of TRIB3 and MYC in Raji cells was detected with immunostaining (PLA assay). Data are representative images from three independent experiments. Scale bar, 2 μm. f Mapping TRIB3 regions binding to MYC. Top: deletion mutants of TRIB3. Bottom: HEK 293T cells were cotransfected with the indicated constructs of TRIB3 (HA tag) and MYC (Flag tag). Cell extracts were IP with an anti-Flag Ab. g, h Mapping MYC regions binding to TRIB3 (g) or UBE3B (h). Top: deletion mutants of MYC. Bottom: HEK293T cells were cotransfected with the indicated constructs of MYC and TRIB3 or UBE3B. Cell extracts were IP with an anti-Flag Ab. i TRIB3 overexpression interfered with the interaction of UBE3B and MYC. HEK 293T cells were cotransfected with the indicated constructs. Cell extracts were IP with an anti-MYC Ab. j TRIB3 decreased the binding of UBE3B and MYC. Kinetic interactions of the UBE3B and MYC proteins were determined by SPR analyses with the BSA or TRIB3 proteins. k TRIB3 overexpression increased the interaction of MAX and MYC. HEK 293T cells were cotransfected with the indicated constructs. Cell extracts were IP with an anti-MYC Ab. l TRIB3 overexpression increased the binding of MAX and MYC. Kinetic interactions of the MAX and MYC proteins were determined by SPR analyses with the CTRL (GST) or TRIB3 (TRIB3-GST) protein. m The heterotrimers of MYC, TRIB3, and MAX in Raji cells were detected by Co-IP assays. Cellular extracts were IP with mouse IgG as a negative control or anti‐MYC, anti‐TRIB3, or anti-MAX antibodies. Western blots were performed with anti‐MYC, anti‐TRIB3, and anti-MAX antibodies. n Recovery of the TRIB3-HA wild-type or KDC-HA mutant enhanced the decreased cell viabilities of TRIB3^Cas9 Jurkat cells for the indicated times. The colors represent different time points; the diameter indicates the relative cell viability. Source data are provided as a Source Data file.