Fig. 5.

TRIM6 promoted TIS21 ubiquitination. a, b, Western blotting (a) and qRT-PCR (b) were used to detect TIS21 in HCT-8 and HCT116 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1, − 2) or control shRNA (shNC). c, HCT-8 cells were transfected with pcDNA3.1-myc-TIRM6 or pcDNA3.1-myc (Vector) for 24 h, and exposed to 20 mM cycloheximide (CHX, Sigma-Aldrich). Cell lysate was prepared at 0, 3 and 6 h after exposure and subjected to western blotting analysis. d, HCT-8 cells were transfected with pCMV-Tag2-TIRM6 or pCMV-Tag2 vector for 24 h and then treated with MG132 (10 μM) or DMSO for 20 h. Western blotting was used to detect TIS21. e, Cell lysates from HCT-8 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1) or control shRNA (shNC) were IP with TIS21-Ab/control IgG and then immunoblotted for ubiquitin (Ub). f, Ubiquitination assay. The 293 T cells were transfected with plasmids expressing myc-TRIM6, His-ubiquitin and FLAG-TIS21 (WT, K5R, K51R or K150R). Cell lysates were incubated with nickelnitrilotriacetic acid beads and subjected to western blotting with anti-FLAG