Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: Spastin is ubiquitinated. (A) Ubiquitin peptides (red font) detected by MS analysis of spastin precipitates from rat brain lysate. (B) GST-ubiquitin was purified and used for GST pulldown of proteins from rat brain lysate. (C) Ubiquitin was immunoprecipitated from rat brain lysate and immunoblotted with an anti-spastin antibody. (D) Spastin was immunoprecipitated with an anti-spastin antibody and immunoblotted with an anti-ubiquitin antibody. (E) Flag-Ub and GFP-spastin were overexpressed in HEK293 cells. The cells were treated with the proteasome inhibitor MG132 (20 µM) for 8 hours, then collected and lysed. GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (F) GFP-Spastin was overexpressed in COS7 cells and HEK293 cells. The cells were treated with CHX (10 µg/mL) for 24 hours and MG132 for 8 hours, and spastin expression was detected. (G) COS7 cells and HEK293 cells were treated with CHX and MG132, and endogenous spastin was detected. (H) HT22 cells were treated with CHX and MG132 and collected at the indicated times for western blotting. Quantification of spastin levels relative to GAPDH is shown. (I) Flag-Ub and Flag-tagged ubiquitin mutants (K48R/K63R) were overexpressed with GFP-spastin in HEK293 cells. Spastin was detected by western blotting. (J) Flag-Ub and Flag-tagged ubiquitin mutants (K48R/K63R) were overexpressed with GFP-spastin in HEK293 cells. Cells were treated with MG132 for 8 hours before collection. Then, GFP-spastin was immunoprecipitated with anti-GFP antibody and immunoblotted with an anti-Flag antibody. n = 3. Data are shown as mean ± SEM. * P < 0.05. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; GST: glutathione-S-transferase; IP: immunoprecipitation; MS: mass spectrum.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Ubiquitin Proteomics, Purification, Immunoprecipitation, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: Spastin ubiquitination sites. (A) Overview of wild-type and truncated spastin. (B) The truncated spastin mutants were expressed in HEK293 cells that were treated with CHX and MG132, followed by western blotting for spastin. (C) The truncated spastin mutants were co-expressed with Flag-Ub in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. Then, GFP was immunoprecipitated with an anti-GFP antibody and immunoblotted with anti-Flag antibody. (D) Potential spastin ubiquitination sites (K219/K386/K484/K497/K519) were predicted using the PhosphoSite tool ( https://www.phosphosite.org/ ). (E) WT or 5KR was expressed in HEK293 cells, which were collected at the indicated times for western blotting. (F) Quantification of spastin levels relative to GAPDH. (G) GFP-labeled wild-type spastin and spastin 5KR were co-expressed with Flag-Ub in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. Then, GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (H) The wild-type and mutant spastin plasmids were transfected into HEK293 cells, which were then treated with CHX. Spastin was detected by western blot at the indicated times. Quantification of spastin levels relative to GAPDH is shown. n = 3. Data are shown as mean ± SEM. * P < 0.05. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; IB: immunoblotting; IP: immunoprecipitation; spastin 5KR: spastin with all five sites mutated; WT: wild-type.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Phospho-proteomics, Labeling, Mutagenesis, Transfection

Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: Cullin3 mediates spastin ubiquitination. (A) Cullin (Cullin3/Cullin4b/Cullin5) peptides were detected by MS analysis of spastin precipitates from rat brain lysates. (B) Flag-tagged Cullins (Cullin3/Cullin4b/Cullin5) were co-expressed with GFP-spastin in HEK293 cells. GFP-spastin expression was detected for western blotting. (C) Increasing amounts of Flag-Cullin3 were co-expressed with GFP-spastin in HEK293 cells, and GFP-spastin expression was detected. (D) Flag-Cullin3 was co-expressed with GFP-spastin in HEK293 cells for 24 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (E) HA-Cullin3 was co-expressed with GFP-spastin in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody, and ubiquitinated spastin was detected by immunoblotting using an anti-Flag antibody. (F) GFP-spastin and Flag-Cullin3 were co-expressed in HEK293 cells that were then treated with CHX. The cells were collected at the indicated times for western blotting. The lysates were blotted with anti-GFP and anti-Flag antibodies. n = 3. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; HA: hemagglutinin; IP: immunoprecipitation.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: USP14 deubiquitinates and stabilizes spastin. (A) USP14 peptides were detected by MS analysis of spastin precipitates from rat brain lysate. (B) Increasing amounts of GFP-USP14 were co-expressed with Flag-spastin in HEK293 cells, and Flag-spastin expression was detected. (C, D) GFP-USP14 and Flag-spastin were co-expressed in HEK293 cells that were then treated with CHX. The cells were collected at the indicated times for western blotting. The lysates were collected and blotted with anti-Flag and anti-GFP antibodies. (E) Flag-USP14 was co-expressed with GFP-spastin in HEK293 cells for 24 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (F) Flag-spastin was co-expressed with GFP-USP14 in HEK293 cells for 24 hours before collection. GFP-USP14 was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (G) HA-USP14 was co-expressed with GFP-spastin in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody, and ubiquitinated spastin was detected with an anti-Flag antibody. n = 3. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; IB: immunoblotting.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 interacted with Snail. (A) UBR5 was positively correlated with Snail in colon adenocarcinoma and rectal adenocarcinoma. Correlation analysis for TCGA and GTEx on the GEPIA website showed a correlation coefficient of R = 0.26, P = 4.4e-13. P < 0.01 denoted statistical significance. (B) The expression levels of UBR5 and Snail correlated with colorectal cancer (CRC) stages. The UBR5 and Snail mRNA levels based on pathological stages were analyzed using the GEPIA2 violin-plots in colorectal tumors. (C) Co-immunoprecipitation assay showed that UBR5 interacted with Snail. HEK293T cells were transfected with Myc-tagged UBR5 and Flag-tagged Snail and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (D) Co-localization of UBR5 and Snail in the nucleus. Immunofluorescence assay probe co-localization of UBR5 (red) and Snail (green). Scale bar: 50 μm. (E) UBR5 interacted with Snail through the HECT domain. A schematic of various UBR5 truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various UBR5 truncations. (F) Snail interacted with UBR5 through the zinc-figure domain. A schematic of various Snail truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various Snail truncations. (G) Molecular docking of Snail zinc-figure domain (amino acids 151–264) and UBR5 HECT domain (amino acids 2453–2799) truncation protein. (H) The HECT domain of UBR5 interacted with Snail in the co-immunoprecipitation assay. HEK293T cells were transfected with wild-type and truncated Myc-tagged UBR5, as well as Flag-tagged Snail, and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail or anti-UBR5 antibodies.

Article Snippet: After 16 h, transfected cells were treated with 10 μM MG132, 20 μM chloroquine (Medchemexpress, HY-17589) for 8 h, or 10 μM CT99021 (Aladdin, C408766) for 24 h. Following treatments, cells were lysed using ice-cold whole-cell extraction buffer (25 mM β-glycerophosphate, pH 7.3, 2 mM EGTA, 10 mM EDTA, 10 mM β-mercaptoethanol, 0.1 M NaCl, 1% Triton X-100, and a protease inhibitor cocktail), and protein lysates were prepared for western blotting analysis.

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Immunofluorescence, Staining

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Article Snippet: After 16 h, transfected cells were treated with 10 μM MG132, 20 μM chloroquine (Medchemexpress, HY-17589) for 8 h, or 10 μM CT99021 (Aladdin, C408766) for 24 h. Following treatments, cells were lysed using ice-cold whole-cell extraction buffer (25 mM β-glycerophosphate, pH 7.3, 2 mM EGTA, 10 mM EDTA, 10 mM β-mercaptoethanol, 0.1 M NaCl, 1% Triton X-100, and a protease inhibitor cocktail), and protein lysates were prepared for western blotting analysis.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Concentration Assay, Ubiquitin Proteomics, Immunoprecipitation