Journal: Neoplasia (New York, N.Y.)

Article Title: SCF β-TrCP targets Ajuba for degradation in a GSK3β-dependent manner in colorectal cancer

doi: 10.1016/j.neo.2025.101175

Figure Lengend Snippet: GSK3β promotes ubiquitination and degradation of Ajuba. A Western blot analysis of HCT116 cells treated with CK1 inhibitor D 4476 (50 μM), GSK3β inhibitor CHIR-99021 (50 μM) and S6K1 inhibitor PF-4708671 (50 μM) for 24 h. B HCT116, HT29 and SW480 cells were incubated with GSK3β inhibitor CHIR-99021 (50 μM) for 24 h and collected for western blot. C Western blot analysis for HCT116 cells treated with 50 μM CHIR-99021 at different time points. D HCT116 cells cultured with GSK3β inhibitor CHIR-99021 (50 μM) for 24 h were pretreated with MG132 (20 μM) for 5 h and harvested for IP. E Western blot analysis of HCT116, SW480 and RKO cells transfected with siRNAs targeting GSK3β for 72 h. F Overexpression of Myc-GSK3β and HA-Ajuba to evaluate the expression level of exogenous Ajuba. G HEK293T cells were transfected with increasing amounts of Myc-GSK3β plasmid, followed by western blot analysis. H, I Cells were transfected with the indicated plasmids for 36 h with or without CHIR-99021 (50 μM) for 24 h and exogenous interaction between GSK3β and Ajuba was analyzed with western blot. J, K Upper panel: RKO cells transfected with two GSK3β siRNAs for 72 h were treated with CHX (50 mg/mL) for the indicated times and then were subjected to western blot. Lower panel: Quantification of Ajuba band intensity. L RKO cells were transfected with GSK3β siRNAs and pretreated with MG132 (20 μM) for 5 h. The cell lysates were immunoprecipitated and then subjected to western blot to analyze the ubiquitination level of Ajuba. M GSK3β and β-TrCP siRNAs were either cotransfected or transfected alone into HCT116 cells and the protein level of Ajuba was detected by western blot.

Article Snippet: CK1 inhibitor D 4476 (S7642), GSK3β inhibitor CHIR-99021 (S1263) and S6K1 inhibitor PF-4708671 (S2163) were purchased from Selleck.

Techniques: Ubiquitin Proteomics, Western Blot, Incubation, Cell Culture, Transfection, Over Expression, Expressing, Plasmid Preparation, Immunoprecipitation