Journal: Cancer Science
Article Title: PSMD12 Overexpression Promotes Lung Adenocarcinoma Progression via Ubiquitin–Proteasome Pathway Dysregulation
doi: 10.1111/cas.70290
Figure Lengend Snippet: PSMD12 regulates the ubiquitination level of CDK1 in lung adenocarcinoma (LUAD) cells. (A) Correlation between PSMD12 and CDK1 protein expression levels in the Clinical Proteomic Tumor Analysis Consortium (CPATC) LUAD dataset ( N = 215); r represents the Pearson's correlation coefficient. (B) Western blot analysis of CDK1 expression in PSMD12‐knockout (upper panel) and PSMD12‐overexpressing (lower panel) cells. The right panels show densitometric quantification of CDK1 protein levels normalized to β‐actin. (C) Cycloheximide (CHX) chase assays in PSMD12‐knockout (upper panels) and PSMD12‐overexpressing (lower panels) PC9 cells. Left panels show representative western blots of CDK1 expression at the indicated time points, and right panels show densitometric quantification of relative CDK1 levels used to assess protein stability. (D) Western blot images of CDK1 expression in PSMD12‐knockout PC9 cells treated with or without the proteasome inhibitor, MG132 (15 μM, 6 h). Quantified CDK1 protein levels are shown in the lower panel. (E) Detection of ubiquitinated CDK1 in control and PSMD12‐overexpressing PC9 cells. Ubiquitinated proteins were immunoprecipitated using ubiquitin antibody–conjugated beads (Signal‐Seeker Ubiquitin Detection Kit), followed by western blotting with an anti‐CDK1 antibody. Densitometric quantification of ubiquitinated CDK1 is shown on the right. (F) Gene set enrichment analysis (GSEA) of The Cancer Genome Atlas LUAD dataset showing that high PSMD12 expression is strongly associated with enrichment of the KEGG_UBIQUITIN_MEDIATED_PROTEOLYSIS pathway. * p < 0.05; ** p < 0.01; *** p < 0.005.
Article Snippet: Ubiquitination levels of CDK1 were evaluated using a Signal‐Seeker Ubiquitination Detection Kit (Cytoskeleton Inc.) according to the manufacturer's instructions.
Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Knock-Out, Control, Immunoprecipitation
