proteasome inhibitor mg132  (TargetMol)

Journal: MedComm

Article Title: TRIM28 Increases RFC4 Protein Expression to Promote Triple‐Negative Breast Cancer Progression

doi: 10.1002/mco2.71043

Figure Lengend Snippet: TRIM28 ubiquitinates RFC4 with K29‐linked polyubiquitin chains. (A) MDA‐MB‐231 cells were transduced with lentiviruses carrying empty vector or oeTRIM28 and treated with cycloheximide (CHX) for 0, 4, 8, and 12 h. (B) TRIM28 knockdown promoted RFC4 protein degradation through the proteasome pathway. (C) Analysis of the ubiquitin chain types on RFC4 affected by TRIM28. Ubiquitinated proteins were pulled down using anti‐Flag agarose under denaturing conditions from HEK293T cells transfected with the indicated constructs and analyzed by western blotting with the indicated antibodies. At 24 h after transfection, cells were treated with MG132 (10 µM) for an additional 4 h. (D) Analysis of endogenous RFC4 ubiquitination in MDA‐MB‐231 cells following siRNA‐mediated TRIM28 knockdown. (E) TRIM28‐mediated K29‐linked polyubiquitination of RFC4. (F) Effect of mutation of the TRIM28 enzymatic active site on the half‐life of RFC4. (G) Quantitative analysis of RFC4 protein levels after mutation of the TRIM28 enzymatic active site. (H) TRIM28 ubiquitinated RFC4 at K205 and K217. (I–K) Analysis of the half‐lives of Flag‐RFC4 wild‐type, K205R, K217R, and K205/217R mutant proteins in HEK293T cells.

Article Snippet: The proteasome inhibitor MG132, protein synthesis inhibitor CHX, autophagy inhibitor CQ and PI3K inhibitor LY294004 were purchased from MedChem Express (Shanghai, China).

Techniques: Transduction, Plasmid Preparation, Knockdown, Analysis, Ubiquitin Proteomics, Transfection, Construct, Western Blot, Mutagenesis