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The SCF E3 ligase complex enhances the ubiquitination of Ajuba. A HCT116 cells were transfected with siRNAs targeting 6 culllins for 72 h and then were subjected to western blot. B HCT116 cells were transfected with <t>CUL1</t> siRNAs for 72 h and then were lysed for western blot analysis. C-E HEK293T cells were cotransfected with indicated plasmids for 36 h and pretreated with MG132 (20 μM) for 5 h before harvest. Lysates were incubated with anti-Flag agarose beads, followed by western blot analysis. F-M Left panel: Cells transfected with siRNAs targeting CUL1, ROC1 and SKP1 were treated with CHX (50 mg/mL) at the indicated time points before harvest and the protein expression of Ajuba was detected by western blot. Right panel: Quantification of Ajuba band intensity. N Immunoprecipitation (IP) and western blot were performed on HCT116 cells transfected with CUL1 siRNAs to determine the ubiquitination level of Ajuba. O HCT116 cells were cultured with DMSO or MLN4924 (1 µM) for 24 h and MG132 (20 μM) for 5 h. The protein extracts were immunoprecipitated with anti-Ajuba antibody, followed by western blot analysis.
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The SCF E3 ligase complex enhances the ubiquitination of Ajuba. A HCT116 cells were transfected with siRNAs targeting 6 culllins for 72 h and then were subjected to western blot. B HCT116 cells were transfected with <t>CUL1</t> siRNAs for 72 h and then were lysed for western blot analysis. C-E HEK293T cells were cotransfected with indicated plasmids for 36 h and pretreated with MG132 (20 μM) for 5 h before harvest. Lysates were incubated with anti-Flag agarose beads, followed by western blot analysis. F-M Left panel: Cells transfected with siRNAs targeting CUL1, ROC1 and SKP1 were treated with CHX (50 mg/mL) at the indicated time points before harvest and the protein expression of Ajuba was detected by western blot. Right panel: Quantification of Ajuba band intensity. N Immunoprecipitation (IP) and western blot were performed on HCT116 cells transfected with CUL1 siRNAs to determine the ubiquitination level of Ajuba. O HCT116 cells were cultured with DMSO or MLN4924 (1 µM) for 24 h and MG132 (20 μM) for 5 h. The protein extracts were immunoprecipitated with anti-Ajuba antibody, followed by western blot analysis.
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The SCF E3 ligase complex enhances the ubiquitination of Ajuba. A HCT116 cells were transfected with siRNAs targeting 6 culllins for 72 h and then were subjected to western blot. B HCT116 cells were transfected with CUL1 siRNAs for 72 h and then were lysed for western blot analysis. C-E HEK293T cells were cotransfected with indicated plasmids for 36 h and pretreated with MG132 (20 μM) for 5 h before harvest. Lysates were incubated with anti-Flag agarose beads, followed by western blot analysis. F-M Left panel: Cells transfected with siRNAs targeting CUL1, ROC1 and SKP1 were treated with CHX (50 mg/mL) at the indicated time points before harvest and the protein expression of Ajuba was detected by western blot. Right panel: Quantification of Ajuba band intensity. N Immunoprecipitation (IP) and western blot were performed on HCT116 cells transfected with CUL1 siRNAs to determine the ubiquitination level of Ajuba. O HCT116 cells were cultured with DMSO or MLN4924 (1 µM) for 24 h and MG132 (20 μM) for 5 h. The protein extracts were immunoprecipitated with anti-Ajuba antibody, followed by western blot analysis.

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: The SCF E3 ligase complex enhances the ubiquitination of Ajuba. A HCT116 cells were transfected with siRNAs targeting 6 culllins for 72 h and then were subjected to western blot. B HCT116 cells were transfected with CUL1 siRNAs for 72 h and then were lysed for western blot analysis. C-E HEK293T cells were cotransfected with indicated plasmids for 36 h and pretreated with MG132 (20 μM) for 5 h before harvest. Lysates were incubated with anti-Flag agarose beads, followed by western blot analysis. F-M Left panel: Cells transfected with siRNAs targeting CUL1, ROC1 and SKP1 were treated with CHX (50 mg/mL) at the indicated time points before harvest and the protein expression of Ajuba was detected by western blot. Right panel: Quantification of Ajuba band intensity. N Immunoprecipitation (IP) and western blot were performed on HCT116 cells transfected with CUL1 siRNAs to determine the ubiquitination level of Ajuba. O HCT116 cells were cultured with DMSO or MLN4924 (1 µM) for 24 h and MG132 (20 μM) for 5 h. The protein extracts were immunoprecipitated with anti-Ajuba antibody, followed by western blot analysis.

Article Snippet: The antibodies used in this study were as follows: anti-Ajuba (A22039) antibody for WB (ABclonal Technology); anti-β-actin (EM21002) and anti-LC3B (ET1701-65) antibodies (HUABIO); anti-Flag-tag (20543-1-AP) antibody (Proteintech); anti-HA-tag (M180-3) antibody (MBL); anti-β-TrCP (4394) for WB, anti-Ajuba (4897) for IF, anti-p27 (3686), anti-GSK3β (12456), anti-SKP1 (12248), anti-Ub (43124) and anti-Myc-tag (2276) antibodies (Cell Signaling Technology); anti-ROC1 (ab133565) antibody (abcam); anti-normal mouse IgG (sc-2025), anti-Ajuba (sc-398008) for IP, anti-β-TrCP (sc-390629) for IF, anti-CUL1 (sc-17775) antibodies (Santa Cruz Biotechnology); anti-Ajuba (HPA006171) antibody for IHC (Merck).

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Incubation, Expressing, Immunoprecipitation, Cell Culture