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

Journal: The Journal of Clinical Investigation

Article Title: A haploinsufficiency restoration strategy corrects neurobehavioral deficits in Nf1 +/– mice

doi: 10.1172/JCI188932

Figure Lengend Snippet: ( A ) Schematic map of the neurofibromin (NF1) interaction domains used to test interactions with FBXW11. Full-length NF1 was divided into the 6 indicated domains and subcloned into GFP-expressing plasmids. Flag-tagged FBXW11 and the various GFP-tagged NF1 subdomains were transfected into HEK293T cells. Six hours prior to harvesting, the cells were treated with 15 μM MG-132. Anti-Flag IP followed by immunoblotting was used to detect GFP-tagged NF1 co-IP with FBXW11. Relative binding activity as determined by immunoblotting is indicated. Image created in BioRender. Angus, S. (2025) https://BioRender.com/n28p603 ( B ) HEK293T cells were treated as in A to confirm the specific interaction of FBXW11 with the domain 3 (D3) fragment of the NF1 peptide, which encompasses the GRD of NF1 isoform 1 (GRD1), but not GRD2. ( C ) A NanoBiT complementation assay was performed after cotransfection of HEK293T cells with LgBiT-FBXW11 and SmBiT-GRD1 or GRD2 fusion proteins. Forty-five hours after transfection, the interaction was detected by luminescence. The negative control (neg. cont.) refers to luminescence values obtained from wells containing cells with LgbiT-FBXW11 and SmBiT-empty. Vehicle or the proteasome inhibitor bortezomib (1 mM) was included to enhance the interaction due to GRD1 accumulation. * P < 0.05 and *** P < 0.001, by unpaired 2-tailed t test. Data indicate the mean ± SEM. Image created in BioRender. Angus, S. (2025) https://BioRender.com/e61l197 ( D ) The SCF complex (SKP1, CUL1, RBX1, and Flag-FBXW11) was purified after cotransfection and subsequent Flag-IP (and Flag-peptide elution) from HEK293T cell lysates. Isolated GFP-GRD1 was incubated at 37°C for 60 minutes in the presence of E1 (100 nM), E2 (2 mM), Mg-ATP (5 mM), and ubiquitin (Ub) in the presence (+) or absence (–) of SCF FBXW11 . Samples were then subjected to immunoblotting, and GRD1 ubiquitination was detected by GFP antibody and the smearing due to higher-molecular-weight species. ( E ) HEK293T cells were transfected with control or FBXW11 -targeting siRNAs for 72 hours. A TUBEs assay was performed using agarose-TUBE2 to pull down total ubiquitinated protein. Immunoblotting was used to detect ubiquitin and NF1.

Article Snippet: The following primary antibodies were used: neurofibromin/NF1 (sc-67, sc-376886, Santa Cruz Biotechnology and ab238142, Abcam); Flag (F7425, MilliporeSigma); GFP (ab1218, Abcam, Western blotting and Invitrogen, IP); turboGFP (AB513, Evrogen); FBXW11/bTrCP2 (GTX33193, GeneTex and PA5-109715, Invitrogen, Thermo Fisher Scientific); ERK1/2 (9102, Cell Signaling Technology); anti-pERK Thr202/Tyr204 (4377, Cell Signaling Technology); IκBα (9242 Cell Signaling Technology); β-catenin (8480, Cell Signaling Technology); ubiquitin (3933, Cell Signaling Technology); CUL1 (17775, Cell Signaling Technology); SKP1 (2156, Cell Signaling Technology); Rbx1 (4397, Cell Signaling Technology); and GAPDH (2118, Cell Signaling Technology).

Techniques: Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Activity Assay, Cotransfection, Negative Control, Purification, Isolation, Incubation, Ubiquitin Proteomics, Molecular Weight, Control