Fig. 7: TRIM25 cooperates with UBE2J1 to enhance the poly-ubiquitination and degradation of RPS3 and the TRIM25/UBE2J1-RPS3 axis suppresses NF-κB signaling pathway.

A The endogenous interaction of UBE2J1 and TRIM25 was tested by co-IP and western blotting assay in DLD-1 and LoVo cells. B HEK-293T cells were transfected with indicated plasmids for 24 h. And the exogenous interaction between UBE2J1 and TRIM25 was detected by co-IP and western blotting assay. C, D Western blotting assay was used to detect the effect of TRIM25 cooperating with UBE2J1 on regulating RPS3 expression. E, F The effect of TRIM25 cooperating with UBE2J1 on poly-ubiquitination of RPS3. HEK-293T cells expressing the indicated plasmids for 24 h were treated with MG132 for 8 h (10 μM). Cell lysates were immunoprecipitated (IP) with an anti-Myc antibody followed by immunoblotting against indicated antibodies. G, H Western blot analysis of p‑P65 and P65 protein levels from whole‑cell, nuclear, and cytoplasmic extracts in DLD-1 and HCT 116 cells stably transfected with the indicated lentiviruses. GAPDH, α-Tubulin, and Lamin B1 served as a loading control. I, J P65 transcription factor DNA binding activity assay in nuclear extracts obtained from DLD-1 and HCT 116 cells stably transfected with the indicated lentiviruses. All data are presented as the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001.