Research Paper Volume 13, Issue 4 pp 5034—5054

Figure 5. TRIM29 negatively regulates PKM1 by promoting its degradation via ubiquitination. (A) A coimmunoprecipitation (Co-IP) assay was performed to identify the proteins in DLD-1 cells that interact with TRIM29. (B) The Co-IP experiments showed that TRIM29 interacted with PKM1 in DLD-1 and SW480 cell lines. (C, D) qPCR and Western blotting were used to measure the mRNA and protein expression levels of PKM1 after overexpressing and knocking down TRIM29. (E) Cells were transfected with vector and TRIM29 plasmid and followed by 100 μM cycloheximide (CHX). The expression levels of PKM1 were examined by Western blotting every 2 h in SW480 cells and every 4 h in DLD-1 cells. The degradation rate of PKM1 is shown in the right chart. (F–G) Cells were transfected with vector or TRIM29 plasmid for 36 h and treated with 10 μM MG132 (a proteasome inhibitor) for an additional 12 h. (F) PKM1 protein levels were examined in SW480 and DLD-1 cells. (G) Co-IP assays were performed. The levels of polyubiquitinated (Poly-Ub) PKM1 proteins was examined by Western blot with anti-ubiquitin antibody in SW480 and DLD-1 cells. (H) The levels of wild type, K48O and K48R polyubiquitinated (Poly-Ub) PKM1 proteins was examined by Western blot with anti-ubiquitin antibody in SW480 cells. The statistical analysis was performed using Kendal’s tau-b test. *P < 0.05, **P < 0.01, ***P < 0.001. Every experiment repeated at least 3 times.